Suppressor of cytokine signaling (SOCS)-3 promoter and methods for its use in genetic therapy in humans

ABSTRACT

Disclosed is a nucleic acid construct comprising a murine SOCS-3 promoter sequence having SEQ. ID. NO.: 1, or a non-murine homologue thereof, or an operative fragment or derivative. The construct can also contain, operatively linked to the SOCS-3 promoter, a gene encoding any preselected protein, and optionally contains a reporter gene to facilitate detection and/or selection of successfully transfected cells. Also disclosed are a transgenic vertebrate cell containing the nucleic acid construct and transgenic non-human vertebrates comprising such cells. The nucleic acid construct is useful in methods of treating a growth retardation or growth acceleration disorder in a human subject and in a method of treating an autoimmune disease, immune disease, or inflammatory condition in a human subject. A kit for genetically modifying a vertebrate cell includes a polynucleotide comprising the murine SOCS-3 promoter sequence is also disclosed.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grant DK50238 awarded by the NIH.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced withinparentheses. The disclosures of these publications in their entiretiesare hereby incorporated by reference in this application in order tomore fully describe the state of the art to which this inventionpertains. 1. The Field of the Invention

This invention relates to the medical arts. In particular the presentinvention relates to the field of cellular signal transduction and togene therapy.

2. Discussion of the Rekated Art

Cytokines are small secreted proteins or factors (5 to 20 kD) that havespecific effects on cell-to-cell interactions, intercellularcommunication, or the behavior of other cells. Cytokines involved ininflammatory diseases are produced by lymphocytes, especially T_(H)1 andT_(H)2 lymphocytes, monocytes, intestinal macrophages, granulocytes,epithelial cells, and fibroblasts. (Reviewed in G. Rogler and T. Andus,Cytokines in inflammatory bowel disease, World J. Surg. 22(4):382-89[1998]; H. F. Galley and N. R. Webster, The immuno-inflammatory cascade,Br. J. Anaesth. 77:11-16 [1996]). Some cytokines are pro-inflammatory(e.g., tumor necrosis factor [TNF]-α, interleukin [IL]-1 (αand β), IL-6,IL-8, IL-12); others are anti-inflammatory (e.g., IL-1 receptorantagonist [IL-1ra], IL-4, IL-10, IL-11, and transforming growth factor[TGF]-β). However, there may be overlap and functional redundancy intheir effects under certain inflammatory conditions.

One group of cytokines, the IL-6-type, are also important in theregulation of complex cellular processes such as gene activation,proliferation and differentiation. The IL-6-type cytokines include IL-6,IL-11, leukemia inhibitory factor (LIF), oncostatin M, ciliaryneutrophic factor, and cardiotrophin-1. (Reviewed in P. C. Heinrich etal., Interleukin-6-type cytokine signaling through the gp130/JAK/STATpathway, Biochem. J. 334(Pt 2):297-314 [1998]). The IL-6-type cytokines(also known as the gp130 signaling subunit cytokine family) have incommon that signal transduction proceeds through a pathway beginningwith ligand binding by type I and type II surface receptors,internalization involving affinity converter/signal transducing subunitgp130, the activation of the Janus family of cytoplasmic tyrosinekinases (e.g., Jak1, Jak2, and Tyk2); this results in thephosphorylation and dimerization of the signal transducers andactivators of transcription (STAT)-1 and STAT-3 that activatetranscription from promoters having STAT recognition sites. (Heinrich etal. [1998]; M. Ernst et al., Gp130-mediated signal transduction inembryonic stem cells involves activation of Jak andRas/mitogen-activated protein kinase pathways, J. Biol. Chem.271(47):30136-43 [1996]; R. Starr et al., A family of cytokine-inducibleinhibitors of signaling, Nature 387(6636):917-21 [1997]; T. Hirano etal., Cytokine & Growth Factor Rev. 8:241-52 [1997]; E. Arzt & G. K.Stalla, Neuroimmunomodulation 3:28-34 [1996]; S. J. Haque & B. R. G.Williams, Semin. Oncol. 25 (suppl. 1):14-22 [1998]). This pathway isknown as the Jak-STAT signaling cascade.

Several IL-6-type cytokines are important neuro-immuno-endocrinemodulators of the hypothalamo-pituitary-adrenal (HPA) axis (Arzt, E. &Stalla [1996]; S. Melmed, Trends Endocrinol. Metab. 8:391-97 [1997]; H.O. Besedovsky, & A. Del Rey, Endo. Rev. 17:64-102 [1996]), whichregulates metabolism, including growth, body temperature, water balance,blood sugar, fat metabolism, and sexual and nerve function. For example,LIF is a potent auto-paracrine stimulus of pituitary proopiomelanocortin(POMC) gene expression and adrenocorticotrophic hormone (ACTH)secretion, which stimulates the adrenals to produce additional hormones.Thus, LIF modulates the HPA axis response to various inflammatory andstress stimuli. (Z. Wang et al., Endocrinology 137:2947-53 [1996]; C. J.Auernhammer et al., Endocrinology 139:2201-08 [1998a]). In vitroexperiments using human fetal pituitary cells (I. Shimon et al., J.Clin. Invest. 100: 357-63 [1997]) and the corticotroph cell line AtT-20(S. Akita et al., J. Clin., Invest 95, 1288-1298 [1995]; C. Bousquet etal., J. Biol. Chem 272:10551-57 [1997]), showed a profound andsynergistic action of LIF and corticotropin-releasing hormone (CRH) onPOMC gene expression and ACTH secretion. LIF is known to induce theJak-STAT signaling cascade in the corticotroph cells. (C. J. Auernhammeret al., Pituitary corticotroph SOCS-3: novel intracellular regulation ofleukemia-inhibitory factor-mediated proopiomelanocortin gene expressionand adrenocorticotropin secretion, Mol. Endocrinol. 12(7):954-61[1998b]; I. Shimon et al. [1997]; D. W. Ray et al., Leukemia inhibitoryfactor (LIF) stimulates proopiomelanocortin (POMC) expression in acorticotroph cell line. Role of STAT pathway, J. Clin. Invest.97(8):1852-59 [1996]; D. W. Ray et al., Ann. N.Y. Acad. Sci. USA840:162-73 [1998]).

A new family of cytokine-inducible proteins has recently been describedthat inhibits the Jak-STAT signaling cascade. (E.g., S. E. Nicholson etal., The SOCS proteins: a new family of negative regulators of signaltransduction, J. Leukoc. Biol. 63(6):665-68 [1998]; R. Starr et al.,SOCS: suppressors of cytokine signaling, Int. J. Biochem. Cell. Biol.30(10):1081-85 [1998]). These proteins have been variously termedsuppressors of cytokine signaling (“SOCS”)(R. Starr et al., A family ofcytokine-inducible inhibitors of signaling, Nature 387(6636):917-21[1998]; D. J. Hilton et al., Proc. Natl. Acad. Sci. USA 95:114-19[1998]), STAT-induced STAT inhibitors (SSI)(T. Naka et al., Nature387:924-28 [1997]; S. Minamoto et al., Biochem. Biophys. Res. Commun237:79-83 [1997]), cytokine-inducible SH2 containing protein (CIS)(A.Yoshimura et al., EMBO J. 14:2816-26 [1995]; M. Masuhara et al.,Biochem. Biophys. Res,. Commun. 239:439-46 [1997]; A, Matsumoto et al.,Blood 89:3148-54 [1997]), and Jak binding protein (JAB)(T. A. Endo etal., Nature 387:921-24 [1997]; H. Sakamoto et al., Blood 92:1668-76[1998]). The SOCS-protein family currently consists of CIS and SOCS-1through 7. (D. J. Hilton et al. [1998]; M. J. Aman & W. J. Leonard,Curr. Biol. 7:R784-R788 [1997]; R. Starr & D. J. Hilton, Int. J.Biochem. Cell Biol. 30:1081-85 [1998]).

SOCS-protein expression is stimulated by various cytokines in a tissuespecific manner (R. Starr et al., Nature 387:917-21 [1997]; M. J. Aman &W. J. Leonard [1997]; H. Sakamoto et al. [1998]; H. O. Besedovsky, & A.Del Rey [1996]; T. E. Adams et al., J. Biol. Chem. 273:1285-87 [1998];C. Bjorbaek et al., Mol. Cell 1:619-625 [1998]). The gene expression ofSOCS-1/SSI-1/JAB and SOCS-3/SSI-3/CIS-3, referred to herein as SOCS-1and SOCS-3, are induced by IL-6 and LIF in various tissues (R. Starr etal. [1997]; D. J. Hilton et al. [1998]; T. Naka et al. [1997]; S.Minamoto et al. [1997]; M. Masuhara et al. [1997]; A. Matsumoto et al.[1997]; T. A. Endo et al. [1997]). For example, SOCS-3 gene expressionis rapidly induced by LIF in the pituitary in vivo, and in corticotrophAtT-20 cells in vitro. (C. J. Auernhammer et al. [1998b]).

Both, SOCS-1 and SOCS-3 proteins bind to the JH1 domain of Jak-2 andthereby inhibit IL-6-, IL-11-, or LIF-induced tyrosine phosphorylationactivity by Jak-2 of gp130 and STAT-3. (S. Minamoto et al. [1997]; M.Masuhara et al. [1997]; C. J. Auernhammer et al. [1998b]). SOCS-3 isinduced by growth hormone (GH) in the liver, and inhibits GH-induced Spi2.1 promoter activity. (T. E. Adams et al. [1998]). SOCS-3 inhibitsLIF-induced POMC gene expression and ACTH secretion (C. J. Auernhammeret al. [1998b]), thus providing an intracellular negative feedbackregulation of cytokine-induced activation of the HPA-axis. HypothalamicSOCS-3 gene expression is stimulated by leptin, and SOCS-3 inhibitsleptin-induced signal transduction (C. Bjorbaek et al., Mol. Cell1:619-625 [1998]), thus suggesting its regulatory role in central leptinresistance.

The structure of SOCS proteins has been described. (e.g., S. E.Nicholson et al., Mutational analyses of the SOCS proteins suggest adual domain requirement but distinct mechanisms for inhibition of LIFand IL-6 signal transduction, EMBO J. 18(2):375-85 (January 1999).Dominant negative STAT-3 mutants, isolated by substitution of acarboxy-terminal tyrosine phosphorylation site Tyr⁷⁰⁵ to Phe⁷⁰⁵(STAT-3F) or mutation at positions important for DNA binding (STAT-3D)have been recently described (K. Nakajima et al., EMBO J. 15:3651-58[1996]). Overexpression of these STAT-3 dominant negative mutants incorticotroph AtT-20 cells inhibits LIF-induced POMC gene expression andACTH secretion. (C. Bousquet & S. Melmed, J. Biol. Chem. 274:10723-30[1999]). Cytokine-induced gene expression of SOCS-1 has been shown to beinhibited in cells overexpressing dominant negative STAT-3 mutants (T.Naka et al. [1997]), but the promoter region of SOCS-1 has not beencloned.

Therefore, there remains a definite need for a promoter sequence capableof regulating expression of preselected proteins, such as SOCS-3protein, and that can be targeted by gene therapy to treat growthdisorders, autoimmune diseases, immune diseases, and inflammatoryconditions. This and other features and benefits provided by the presentinvention will now be described.

SUMMARY OF THE INVENTION

The present invention relates to a nucleic acid construct comprising amurine SOCS-3 promoter sequence, or a non-murine homologue thereof, oran operative fragment or derivative of any of these. The construct canalso contain, operatively linked to the SOCS-3 promoter, a DNA sequenceencoding a gene for any preselected protein or a gene-specific part ofsuch a DNA sequence, or to a DNA sequence that encodes a preselectedgene-specific antisense RNA or a catalytic RNA. A preselected proteinthat is encoded by the nucleic acid construct can be from an autologous,allogeneic, or xenogeneic source. In addition, the present nucleic acidconstruct optionally contains a reporter gene to facilitate detectionand/or selection of successfully transfected cells. The present nucleicacid construct is particularly useful for linking expression of adesired gene product to physiological processes that are regulated bygp130-mediated signal transduction from IL-6-type cytokines (i.e.,cytokines of the gp130 signaling subunit cytokine family), such as IL-6,IL-11, or LIF. For example, when the encoded protein is a SOCS-3protein, the present nucleic acid can be used to modulate the physiologyand/or hormonal secretions of cells of the hypothalamus, pituitary,adrenals, liver, or other tissues, through a negative autoregulatoryfeedback of SOCS-3 on its own cytokine-induced gene expression.

The present invention also relates to a transgenic vertebrate cellcontaining the nucleic acid construct of the present invention and totransgenic non-human vertebrates comprising such cells.

The present invention also relates to a method of treating a growthretardation disorder in a human subject. The method involves geneticallymodifying a GH-responsive or gp130-responsive cell(s) of a human subjecthaving a growth retardation disorder, such as dwarfism, GH deficiency,gonadal dysgenesis, chondrodystrophy, or bone-cartilage dysplasia. Thecell(s) are genetically modified using a nucleic acid construct thatcomprises a SOCS-3 promoter sequence, or operative fragment thereof,operatively linked to a DNA sequence that encodes an RNA thatspecifically hybridizes to a functional SOCS-3 mRNA. In response to agrowth-inducing cytokine, in vivo, the genetically modified cell(s)within the human subject, transcribe an RNA transcript that specificallyhybridizes to a functional SOCS-3 mRNA, preventing translationtherefrom. This RNA transcript can be an antisense RNA or a catalyticRNA (ribozyme) that cleave the SOCS-3 mRNA. As a consequence, the amountof SOCS-3 protein produced within the genetically modified cell(s) isrelatively reduced, and one or more symptoms of the growth retardationdisorder in the subject are thereby improved, due to a lessening ofSOCS-3-mediated signal suppression within the genetically modifiedcell(s).

The present invention also relates to a method of treating a growthacceleration disorder in a human subject. The method involvesgenetically modifying a GH-responsive or gp130-responsive cell(s) of ahuman subject having a growth acceleration disorder, such as gigantism,acromegaly, or Cushing's disease. The cell(s) are genetically modifiedusing a nucleic acid construct, comprising a SOCS-3 promoter sequence,or operative fragment thereof, operatively linked to a DNA sequenceencoding a SOCS-3 protein, or functional fragment thereof. In responseto the growth-inducing cytokine, in vivo, the genetically modifiedcell(s) produce an enhanced amount of SOCS-3 protein. The symptom(s) ofthe growth acceleration disorder in the subject are thereby improved,due to enhanced SOCS-3-mediated cytokine signal suppression.

The present invention also relates to a method of treating an autoimmunedisease, immune disease, or inflammatory condition in a human subjecthaving a condition, such as Crohn's disease, ulcerative colitis,multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis,Grave's disease, or a neuroendocrinological response to psychological orphysical stress. The method involves genetically modifying agp130-responsive cell(s), responsive to a pro-inflammatory cytokine,such as IL-6 or LIF. The cell(s) are genetically modified using anucleic acid construct that includes a SOCS-3 promoter sequence, oroperative fragment thereof, operatively linked to a DNA sequenceencoding a SOCS-3 protein, or functional fragment thereof. In responseto a pro-inflammatory cytokine of the gp130 signaling subunit cytokinefamily, in vivo, the genetically modified cell(s) produce an enhancedamount of SOCS-3 protein. The symptom(s) of the autoimmune disease,immune disease, or inflammatory condition in the subject are therebyimproved, due to a relative increase in SOCS-3-mediated signalsuppression.

Alternatively, the SOCS-3 promoter is operatively linked to a DNAsequence encoding a functional anti-inflammatory cytokine of the gp130signaling subunit cytokine family, such as IL-11, linked to a functionalsecretory signal. In response to a pro-inflammatory cytokine of thegp130 signaling subunit cytokine family, in vivo, the geneticallymodified cell(s) produce and secrete an enhanced amount of theanti-inflammatory cytokine. The symptom(s) of the autoimmune disease,immune disease, or inflammatory condition in the subject are therebyimproved.

The present invention also relates to a kit for genetically modifying avertebrate cell. The kit includes a polynucleotide comprising a murineSOCS-3 promoter sequence having SEQ. ID. NO.:1, or an operative fragmentor non-murine homologue thereof, or an operative derivative of any ofthese. Preferably, the polynucleotide includes, operatively linked tothe SOCS-3 promoter, at least one DNA sequence encoding a preselectedprotein or a gene-specific part of such a DNA sequence, or a DNAencoding a preselected gene-specific antisense RNA or a specificcatalytic RNA, as appropriate for a particular application. Optionally,the promoter is linked to a reporter gene for facilitating detection,isolation, or selection of genetically modified cells from unmodifiedcells. Some embodiments of the kit are configured for use in practicingthe present methods of treating a growth retardation or accelerationdisorder in a human subject or the present method of treating anautoimmune disease, immune disease, or inflammatory condition in a humansubject.

These and other advantages and features of the present invention will bedescribed more fully in a detailed description of the preferredembodiments which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows stimulation of expression from the murine SOCS-3 promoterin corticotroph AtT-20 cells treated with 0.5×10⁻⁹ M LIF, IL-6, or IL-11for 60 and 120 min, respectively. FIG. 1A shows a Northern blot analysisperformed with 25 μg total RNA per lane. The upper panel shows SOCS-3mRNA; the lower panel shows β-actin mRNA. FIG. 1B shows luciferaseactivity in AtT-20 cells transfected with pGL3Basic alone or a−2757/+929 murine SOCS-3 promoter-pGL3Basic construct (clone 6).

FIG. 2 shows LIF-induced SOCS-3 promoter activity and gene expression incorticotroph AtT-20 cells overexpressing wild type STAT-3 (AtT-20W) ordominant negative STAT-3 mutants (AtT-20F and AtT-20D), as well as wildtype SOCS-3 (AtT-20S) and mock-transfected (AtT-20M); cells were treatedwith 0.5×10⁻⁹ M LIF for 45 min. FIGS. 2A and 2D show Northern blotanalysis performed with 15 μg total RNA per lane for a representativeexperiment; upper panel shows SOCS-3 mRNA; lower panel shows β-actinmRNA. FIGS. 2B and 2E show Northern blot signals for SOCS-3 mRNAanalyzed by quantitative densitometry and normalized for β-actin mRNA.FIGS. 2C and 2F show relative luciferase activity in various cell clonesbearing a −2759/+927 murine SOCS-3 promoter-pGL3Basic construct (clone6).

FIG. 3 shows relative luciferase activities in transiently transfectedAtT-20 cells bearing different constructs of the genomic 5′-region ofmurine SOCS-3. Luciferase activity was measured in untreated (filledbars) and LIF-stimulated (unfilled bars) AtT-20 cells. Crossed linesindicate a deletion of STAT binding elements in Clone 6D1(SEQ.ID.NO.:35) and 6D2 (SEQ.ID.NO.:36), in between the namednucleotides. A dotted line indicates a mutation of the wild type STATbinding sequence (5′-TTCCAGGAA-3′; SEQ. ID. NO:13) with mutant(5′-ATCGACGAT-3′; SEQ. ID. NO.:14) in clone 6M1 (SEQ.ID.NO.:37). Clone 2corresponds to SEQ.ID.NO.:38; Clone 4 corresponds to SEQ.ID.NO.:2; Clone6 corresponds to SEQ.ID.NO.:3; Clone 6T1 corresponds to SEQ.ID.NO.:4;Clone 6T2 corresponds to SEQ.ID.NO.:5; Clone 6T3 corresponds toSEQ.ID.NO.:12; Clone 6T4 corresponds to SEQ.ID.NO.:6; and Clone 8corresponds to SEQ.ID.NO.:39. The nucleotide sequence at the top of FIG.3, marked as −101 through −62, corresponds with nucleotide positions2807 through 2846 of SEQ.ID.NO.: 1.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention relates to a nucleic acid construct comprising amurine SOCS-3 promoter sequence, or an operative fragment thereof, or anon-murine homologue thereof, or an operative derivative of any ofthese.

The following nucleotide sequence represents the full length ˜3.8-kbgenomic 5′-region of the murine SOCS-3 gene (GenBank AccessionAF117732). The transcription start site is defined as +1. Anuntranscribed region extends from nucleotide −2907 to −1, inclusive. Atranscribed but untranslated region (exon 1) extends from +1 to +289;exon 2, begins at +854 ( exons are underlined), and contains theintronless coding region of SOCS-3 with a translational start siteencoded at nt. +944 to +946. (R. Starr et al. [1997]). The translationinitiation codon ATG (nt. +944 to +946) and a TATA-box (nt. −39 to −34)are indicated in boldface type. Two potential STAT-binding elements (nt.−74 to −66 and nt. −97 to −88) are in boldface underlined:

−2907 GACGTTCCTA AAAGCATGCA TGTCACCCAG CTTACCCACC CATCTCAGGC CACAGCAGCC−2847 TGAGAGAGCG GAAGAACACC TGCTGGTCCT GTCCCACCTC TCCTCTTCAA ACAGCCCCAC−2787 ATCCTCCAGT TTTGCTCTGG GTGGAGCTCC CTGCTGGCCC TGCAGAGGGA AGGCTCTCCT−2727 AAGCATCATC TATCAGAACG TCTTCAAAAA AAAAAAAAAA AAAAAAAAAG CCTCTCCAGC−2667 CAGGCTAGCT CTAACACCAT TTCTTCCCCT TCCCCTCTCT CAAATTCACT TATCTTTTTT−2607 TTTTTTTTTT TTTTTGGATT TTTGAGACAG GGTTTCTCTG TATAGCCCTG GTTGTCCTGG−2547 AACTCACTTT GTACACCAGC CTGGCCTCGA ACTCAGAGAT CCACCTGCCT CTGCCTCCTG−2487 AGTGCTGGGA TTAAAGGCGT GCGCCACCAC GCCCGGCTAA ATTCACTTAT CTATTTAATG−2427 TATATAGGGT ATAGGCTGCC CTTGAACTCA CAAAGATCTG CTTGCTTTGC TTCTGGAATA−2367 CTAAAGGTGT GTGCTACCAT CACAGGGACC AAGATTTATT TTAATTCTGT ATATGTGTGT−2307 GTGTGTGTGT GTGTATGGGG GGTGCACATG AGTACAGATT CCCTTGGAGG CCTGGGGTGG−2247 CTTAGGACTG GGGTTACAAC AGTTGTGACC CATCCTACAT AGGTCCTGGC ACCAACACCC−2187 CCCCCCCCCC CCCCCGTCTT CCAGAAGTGC AGCAGGTGTT CTTAACTGCT GAGCCAGCAA−2127 TCCAGCCCCT GACTTCCCTC TCTTACTTAA GAAGCTATCA CAGTGTCTCA CTGGGTCACA−2067 ATCATGACTA GTCCTTGCTC ATGGCCCACA GCCTCTTCCC CACTGTGGGT TTTGCCCCGC−2007 AGCTCTGCCG CCCCAGCGCT GCACCCGAGG CCTGACAGAG CCAGGCACGA AGTCAGGGTT−1947 TGTGGAATGG ATGAATGAAC TTGACTCGTG GCAGAGCATT GTAATTTACA AAGCACTTTC−1887 CCATCCATTA ACTCCAGGGC TATTTCCTAA GAGTCCTCCC TGTCCTCCAC TGCCCTCGGC−1827 TCAGAGGCAT ACGGTCAAGG CAGTGGCTGG GGAACACTCC CTGAATGAGA TCAAGGAGGG−1767 CTTGTTCACA GAGAAAGGGA GAATCCATTT GGGGAGCCTG AGAGTGACTC GAAGGCAAGG−1707 ACTGGGCCTC ACCTGTGGGA TCTCCATCTG TGAGCATCCG CTCATCAGAC CAGTGTGAGA−1647 TATTTTAAAT AAGGCCCCTA AGCCTCTTGA CTACTGGAAT TGCCAGGGGC GGGGGACAGA−1587 TGGGCACCCA TCCCTATTTA ACAGATAACA AGACTGAGTC CAGAGAGGCA GTGCACCTGC−1527 CCTGGTCTCT CTTAGTTCCT CAGCATCAGT GGAGCAGATT GGACACAGTG GGCCAGAAGG−1467 GAAGCAGGCA GCCCTCCCTC CTAGCCCAAG CTACTCTGTG TAGTCAGTTT GCCCTCCTAC−1407 TGGTGTTACA AGAAGCCTGT GGTATCCAAG AGGGCAGGTC AGAAAGCCCA CTGAGAGCAG−1347 ACACTGTGTG TCACTTAGCT GGTTCTCAGG TGGCTGCCAC TTCCTGCTGC CTGTTGCAAA−1287 ACTCGACACT AGGCCTTTAT AGATACTCAC GTGACCAGGA GTAAACAACC TTTCACCTCA−1227 ATCACCTGCT CTTATCAATA CTCCCTCTCC ACCCCACCAT CGGGAAAGTT CAGACACCTT−1167 AAAACGTAGA GGCAAGAGAG GGTCCATTCT GACACCTCAG CGACTTTCAG GCAGTGGCTG−1107 AACCCGTTAC AACGCTCTGT GGACAGTCCT CCTAGTCGAC ATTCCTTCTC AGGTTTGACC−1047 CTGTCCTGGG AAGTGAGGCT TCTCTCTCTG GGTTCCCCAC TCCTGTTCTT GAATAAGGAG−987 CCCCACAACC TCTTATTCTC TCTATACAGA GCCTGGGAAA CAGCAAAACT CGGCTCGCCT−927 ACAAGACTCC AGCGCGCCCT CTGGTGGACT CGGGGGACGA GCATGGGATG AGGGTTTCTT−867 TCCTCTAGCT CCCCCACCGC GCCGAGAGTA CCTGGGCGGA CCCACAGTTC GCCACGCAGG−807 TTGGGAGGCC CAGATGAGTG ATAAGGTAGT AGTTAGCTGC TCCTCCCACC CCACTCCCCA−747 AAGGACATCA GCACCCACGT CTGTCACCGA AGAACCAGGC AATGGGCGGA TGAGCTGAGG−687 CCAGGTAGCT GCTTCTAAGT CAGTGTCTCC TCCACTTCTG GATCTCACAG CTTCATCTTT−627 TGGACCTGTC TACAGGTAAA TGTCGCGCAT CCCCCTCCTC CACTTCCTAG GTCCCCAGTG−567 GGCTGGTGGC TGAATGGTCC TACGTCCCTT TTGGTTGGCA CGGGATGCTT GGAACTGTAC−507 ATGAGGACCT CGGGGTGGCC TGGGTGCAGA GGGAGGGGAG CGTCCCCGCG GGGATCAAAA−447 GAAAGGGAAG GGGTGCCAGG AGGGAGCCTC TCCCGGCTGG CCTCCTAGAA CTGCCCGCGC−387 GCTCCCATCG CGACGCCCCC GCCTCTGCCA GAAACCAGCC TTCTTAGAAG GGAGGGGGGG−327 GAAAGTGTGA ATGAGAAGTT GGGGGCGGAG CGCGCGGGGG AGGGGCCGCT GCCAGGAACG−267 CTCGGCCAAG GCTGGCGCCG CGCCCGCCGG TCGGGCAGCC TCGCGCCGCG CTTTGTCTCC−207 CTCTCGGTGA GTCTCGGCGG GTCCTGGAGG CCCCAGCTCC AAGCCCGCCC TCCGCAGCCC−147 CTCCCTCGCC CTCCGCGCAC AGCCTTTCAG TGCAGAGTAG TGACTAAACA TTACAAGAA G−87 ACCGGCCGGG CAG TTCCAGG AA TCGGGGGG CGGGGCGTAC TGGCCGGGTA AATACCCGCG−27 CGCGCGGCCT CCGAGGCGGC TCTAACTCTG ACTCTACACT CGCCCGCTCC TACGACCGCT+34 GTCTCTCCGG GCTCCCGGAC GCCCCCTTCC CGGCCCAGCT CTCCGTCGAG GTCCCTCGCC+94 CAGGTCCTTT GCCTGATTCG CCCAGGAGTG CGCCTCATCG GCCCGGGGAG CAGCGAAGCC+154 AGAGGGGGCG CACGCACGGG GAGCCCCTTT GTAGACTTCA CGGCTGCCAA CATCTGGGCG+214 CAGCGCGAGC CACTGCTGGG CGCCGCCTCG CCTCGGGGAC CATAGGAGGC GCAGCCCCAA+274 GGCCGGAGAT TTCGCT TCGG GACTAGGTAG GAAGGAGGGG CGCGGTGTGG GGAAGGGTGG+334 GGGCATCGGT CCAGCTCGGG AGCTTTTCCC GGTTTCTCCT CCCCTTCCCG GGTCATTCCC+394 GGTAGGGAGG GGACGAGGCA GGGGGCAGAG CGGATGAGAA CCGAAGATCC CTGATTCCCG+454 TCATACTCAG ACTGGGGCCC TCGGGTTTCT CCTGTCCCCT CTCTCACATA TCTCGGGTTT+514 GGCACCCCCC TTTTTTCGCC CTCGCCACTG AGGACACCGG ACTGAGAGGC GCCCTGAGCG+574 TCCCTAGGGC TCTTGTGTCT CTCCCCATCC TGGCCGCGCT CCTGGAGACC CAACTTCCAC+634 GCGCGAGTTT TCTCTGGGCG TCCTCCTAGG GCGGGCAGGG GAAGAGACTG TCTGGGGTTG+694 GCCGGCAGTG ACCGAGGACA GTCGAGTTCC GCGAGGTGGC TGGGCCTGAG ACACGGTCTA+754 AAGCGGGGCA AAGGGGTGCC CCGGGCGCTA GGCGGAGGCT GGAGGGCCGG GCACGCTGGA+814 GGGTTCCGGG CACTCACGCG CCTCACGCTT TGCTCTCTGC AGCTCCCCGG GATGCGGTAG+874 CGGCCGCTGT GCGGAGGCCG CGAAGCAGCT GCAGCCACCG CCGCGCAGAT CCACGCTGGC+934 TCCGTGCGCC ATGGTCACCC ACAGCAAGTT TCCCGCCGCC GGGATGAGCC GCCCCCTGGA+994 CACCAGCCTG CGCCTCAAGA CCTTCAGCTC CAAAAGCGAG// (SEQ. ID. NO.:1)

A preferred embodiment of the SOCS-3 promoter of the present inventionis a DNA fragment with the sequence of nt. −2907 to +1033, inclusive(SEQ. ID. NO.:1). Other preferred embodiments of the SOCS-3 promoterinclude any operative fragment of SEQ. ID. NO.: 1 or non-murinehomologue thereof, or an operative derivative of any of these.

Preferred examples of an operative fragment include the −2759 to +104fragment (SEQ. ID. NO.:2); the −2759 to +927 fragment (SEQ. ID. NO.:3);the −1864 to +927 fragment (SEQ. ID. NO.:4); the −857 to +927 fragment(SEQ. ID. NO.:5); the −63 to +927 fragment (SEQ. ID. NO.:6); the −97 to+927 fragment (SEQ. ID. NO.:7); the −97 to +104 fragment (SEQ. ID.NO.:8); the −87 to +927 fragment (SEQ. ID. NO.:9); the −87 to +104fragment (SEQ. ID. NO.:10); and the −275 to +158 fragment (SEQ. ID.NO.:11). A most preferred example is the −161 to +927 fragment (SEQ. ID.NO.: 12).

Non-murine homologues include any SOCS-3 promoter sequence of non-murineorigin that functions in a vertebrate cell type of interest.

Another preferred embodiment of a SOCS-3 promoter is an operativederivative of SEQ. ID. NO: 1, or of any operative fragment of SEQ. ID.NO.:1 or non-murine homologue thereof, having the translational startsite (the ATG in bold at nt. +944 to +946 of the murine sequence above)changed to ATT, or changed to a codon sequence, other than ATT, that isalso not recognized as a translational start site; another preferredSOCS-3 promoter is a derivative of SEQ. ID. NO.:1 with the codon of thefirst translational start site deleted altogether. Other operativederivatives include SOCS-3 promoter sequences containing a mutation,polymorphism, or variant allele with respect to any nucleotide positionof SEQ. ID. NO.:1 that does not fully eliminate promoter activity, forexample, a deletion of nt. −101 to −62, or a deletion of nt. −80 to −60,or a mutation of nt. −74 to −66. The skilled practitioner is aware ofsuitable methods for site-directed mutagenesis, e.g., the method of Dengand Nickoloff (W. P. Deng and J. A. Nickoloff, Analyt. Biochem.200:81-88[1992]), and commercial site-directed mutagenesis kits are available,for example Transformer® site-directed mutagenesis kit (Clontech).

The murine SOCS-3 gene promoter contains a pair of STAT binding elementsTT(N₅)AA (SEQ. ID. NO.:31), separated by 14 nucleotides, at nt. −74 to−66 and at nt. −97 to −88. In this respect, the murine SOCS-3 promoteris structurally similar to the human CIS gene promoter, which containstwo functionally important pairs of STAT binding elements and isupregulated by a STAT-5 dependent pathway. (A. Yoshimura et al. [1995];A. Matsumoto et al. [1997]; F. Verdier et al., Mol. Cell. Biol.18:5852-60 [1998]). However, for activity from the present SOCS-3promoter, only the STAT binding element at −74 to −66 is essential foroptimal operability.

In a preferred embodiment, the SOCS-3 promoter is operatively linked toa DNA having a DNA sequence encoding any preselected protein or seriesof preselected proteins. For purposes of the present invention,“operatively linked” means that the promoter sequence, is locateddirectly upstream from the coding sequence and that both sequences areoriented in a 5′ to 3′ manner, forming a transcriptional unit, such thattranscription could take place in vitro in the presence of all essentialenzymes, transcription factors, co-factors, activators, and reactants,under favorable physical conditions, e.g., suitable pH and temperature.This does not mean that, in any particular cell, conditions will favortranscription.

These DNA sequence encoding a preselected protein(s), or a gene-specificpart, are derived from the genome of any eukaryotic organism,prokaryotic organism, or virus, and can be autologous, allogeneic, orxenogeneic with respect to the host cell. DNA sequences having a“normal” form of a gene, or a desirable allele thereof are useful ingenetic therapy to compensate for endogenous production of defectiveprotein(s) or the underexpression or overexpression of normalprotein(s). In some embodiments, natural variant alleles of a gene areused, or novel genetic modification(s) are artificially induced in theDNA sequence encoding the preselected protein. Variant alleles ormutations are not limited to single nucleotide polymorphisms (SNPs), butalso include deletions, insertions, inversions, translocations,transitions, tranversions, or repeats. Mutations or variations areartificially induced in the DNA sequence encoding the preselectedprotein by a number of techniques, all of which are well known in theart. Alternatively, the DNA sequence linked to the SOCS-3 promoterencodes a gene-specific antisense RNA, such as an antisense RNA thatspecifically hybridizes to SOCS-3 mRNA, preventing translationtherefrom. In another embodiment, the DNA sequence encodes a catalyticRNA, such as a “hairpin” or “hammerhead” ribozyme, that specificallyhybridizes to a predetermined mRNA of interest and cleaves it, therebypreventing any further translation therefrom.

Most preferably, transcription of the DNA sequence from the SOCS-3promoter results in RNA transcript that is biologically active in thecell or organism of interest, for example, as mRNA that is translatedinto functional protein(s); or as antisense RNA that specificallyhybidizes with a functional mRNA of interest, for example a SOCS-3 mRNA,and thus prevents its translation to protein; or as catalytic RNA thatspecifically hybridizes with and cleaves a predetermined mRNA ofinterest.

In one embodiment, the preselected protein is a SOCS-3 protein, or afunctional fragment thereof. Transcription of the DNA sequence encodingthe SOCS-3 protein produces mRNA transcript, which is translated intoSOCS-3 protein, or a functional fragment thereof. Thus one benefit ofthe present invention is that the nucleic acid can be used in a genetictherapy to correct clinical disorders derived from defective negativeregulation of cytokine signal transduction in GH-responsive orgp130-responsive cells. Such defective negative regulation can resultfrom, but need not result from, endogenous underexpression of functionalSOCS-3 protein, which protein inhibits in an autocrine manner thecytokine-induced Jak-STAT cascade and SOCS-3 protein synthesis itself.But an unmodulated cellular response to GH and IL-6-type cytokinesignaling caused by a defect in any of various components of thecellular signal transduction mechanism can also be negatively regulatedusing the present nucleic acid construct containing a DNA sequenceencoding a SOCS-3 protein, or a functional fragment thereof.

In another embodiment, the DNA sequence operatively linked to the SOCS-3promoter, encodes a SOCS-3-specific nucleotide sequence, transcriptionof which results in the production of RNA transcript in an antisenseorientation that can hybridize to SOCS-3-encoding mRNA to preventsynthesis of SOCS-3 protein. In another embodiment, SOCS-3-specificsequences are included in a DNA sequence that encodes a catalytic RNAthat specifically hybridizes to SOCS-3 mRNA. These embodiments arebeneficially applied to genetic therapy to correct clinical disordersderived from negative overregulation of cytokine signal transduction inGH-responsive or gp130-responsive cells.

Other preferred embodiments of the present nucleic acid construct alsoinclude, operatively linked to the SOCS-3 promoter, a DNA sequenceencoding a reporter protein for facilitating the detection or selectionof cells containing the present nucleic acid construct and expressingfrom the SOCS-3 promoter. Preferably, but not necessarily, the reportergene encodes a fluorescent protein. Fluorescent proteins include greenfluorescent protein (or enhanced green fluorescent protein), yellowfluorescent protein, blue fluorescent protein, a phycobiliprotein, suchas phycoerythrin or phycocyanin, or any other protein which fluorescesunder suitable wave-lengths of light. Another reporter gene suitable forsome applications is a gene encoding a protein that can enzymaticallylead to the emission of light from a substrate(s); for purposes of thepresent invention, such a protein is a “light-emitting protein.” Forexample, a light-emitting protein includes proteins such as luciferaseor apoaequorin.

The DNA of animal cells is subject to methylation at the 5′ carbonposition of the cytidine bases of CpG dinucleotides. Unmethylated CpGsare found preferentially in transcriptionally active chromatin. (T.Naveh-Many et al., Active gene sequences are undermethylated, Proc.Natl. Acad. Sci. USA 78:4246-50 [1981]). Hypermethylation is associatedwith transcriptional repression. (R. Holliday, The inheritance ofepigenetic defects, Science 238:163-70 [1987]). Since some vertebratecell types of interest may silence expression from the present SOCS-3promoter sequence by methylation, the skilled practitioner is aware thatsuitable insulator elements are employed to prevent methylation of thepromoter sequence. Preferably, this is done by flanking thetranscriptional unit of the promoter sequence and included gene(s) withinsulator elements. For example, by including double copies of the 1.2kb chicken β-globin insulator element 5′ to the SOCS-3 promoter sequenceand 3′ to the operatively linked gene(s) in the present DNA construct,methylation will be substantially prevented at CG dinucleotide siteswithin the SOCS-3 promoter sequence and thus expression therefromoccurs. (M. J. Pikaart et al., Loss of transcriptional activity of atransgene is accompanied by DNA methylation and histone deacetylationand is prevented by insulators, Genes Dev. 12:2852-62 [1998]; Chung etal., DNA sequence which acts as a chromatin insulator element to protectexpressed genes from cis-acting regulatory sequences in mammalian cells,U.S. Pat. No. 5,610,053).

The present invention also relates to a transgenic vertebrate cellcontaining the nucleic acid construct of the present invention,regardless of the method by which the construct was introduced into thecell. The present cell is a growth hormone (GH)-responsive orgp130-responsive cell, for example, a cell that specifically binds anyIL-6-type cytokine (i.e., binds a cytokine of the gp130 signalingsubunit cytokine family). Embodiments include pituitary cells,hypothalamic cells, adrenal cells, intestinal cells, kidney cells, livercells (e.g., hepatocytes), immune-competent cells, or bone-formingcells, such as osteoblasts. In one embodiment, the present cell is acorticotroph cell, but the cell may also be an intestinal epithelialcell, a lymphocyte, a somatotroph, a lactotroph, or a gonadotroph cell.For some in vitro applications, for example with a wide variety ofnon-murine cells, inhibitors of histone deacetylation and DNAmethylation, such as trichostatin A or sodium butyrate, can be includedin the culture medium to prevent possible silencing of expression fromthe SOCS-3 promoter. (M. J. Pikaart et al. [1998]).

The transgenic cells of the present invention are detected, isolated orselected from non-transgenic cells with the aid of, for example, aflow-activated cell sorter (FACS), set at the appropriate wavelength(s).Alternatively, the transgenic cells are detected, isolated or selectedmanually from non-transgenic cells using conventional microscopictechnology.

In particular applications involving a transgenic cell that expressesadditional xenogeneic genes from any promoter, this expression may belinked to a reporter gene that encodes a different fluorescent orlight-emitting protein from the reporter gene linked to the SOCS-3promoter. Thus, multiple reporters fluorescing or emitting at differentwavelengths can be chosen and cell selections based on the expression ofmultiple traits can be made.

The present invention also relates to transgenic non-human vertebratescomprising such cells, for example, non-human primates, mice, rats,rabbits, gerbils, hamsters, canines, felines or other non-human mammals.Other vertebrates include birds such as chickens, turkeys, ducks,ostriches, emus, geese, guinea fowl, doves, quail, rare and ornamentalbirds, and the like. Broadly speaking, a “transgenic” vertebrate is onethat has had foreign DNA permanently introduced into its cells. Theforeign gene(s) which (have) been introduced into the animal's cells is(are) called a “transgene(s).” The present invention is applicable tothe production of transgenic vertebrates containing xenogeneic, i.e.,exogenous, transgenic genetic material, or material from a differentspecies, including biologically functional genetic material, in itsnative, undisturbed form. In other embodiments, the genetic material is“allogeneic” genetic material, obtained from different strains of thesame species, for example, from animals having a “normal” form of agene, or a desirable allele thereof.

Gene delivery is by any suitable method including in vivo and vitro genedelivery methods. (E.g., D. T. Curiel et al., U.S. Pat. Nos. 5,521,291and 5,547,932). Typically, gene delivery involves exposing a cell to agene delivery mixture that includes preselected genetic materialtogether with an appropriate vector, mixed, for example, with aneffective amount of lipid transfecting agent (lipofection). The amountof each component of the mixture is chosen so that gene delivery to aspecific species of cell is optimized. Such optimization requires nomore than routine experimentation. The ratio of DNA to lipid is broad,preferably about 1:1, although other proportions may also be utilizeddepending on the type of lipid agent and the DNA utilized. Thisproportion is not crucial. Other well known gene delivery methodsinclude electroporation or chemical methods. (E.g., M. Ostresh, Nobarriers to entry: transfection tools get biomolecules in the door, TheScientist 13(11):21-23 (1999). “Transfecting agent”, as utilized herein,means a composition of matter added to the genetic material forenhancing the uptake of exogenous DNA segment(s) into a vertebrate cell.The enhancement is measured relative to the uptake in the absence of thetransfecting agent. Examples of transfecting agents includeadenovirus-transferrin-polylysine-DNA complexes. These complexesgenerally augment the uptake of DNA into the cell and reduce itsbreakdown during its passage through the cytoplasm to the nucleus of thecell.

Other preferred transfecting agents include Lipofectin®, DMRIE C,Cellfectin® or Lipofectamine (Life Technologies), LipoTAXI (Stratagene),Superfect or Effectene (Qiagen). Although these are not as efficientgene delivery (or transfecting) agents as viral transfecting agents,they have the advantage that they facilitate stable integration ofxenogeneic DNA sequence into the vertebrate genome, without sizerestrictions commonly associated with virus-derived transfecting agents.A virus, or transfecting fragment thereof, can be used to facilitate thedelivery of the genetic material into the cell. Examples of suitableviruses include adenoviruses, adeno-associated viruses, retrovirusessuch as human immune-deficiency virus, other lentiviruses, such asMoloney murine leukemia virus and the retrovirus vector derived fromMoloney virus called vesicular-stomatitis-virus-glycoprotein(VSV-G)-Moloney murine leukemia virus, mumps virus, and transfectingfragments of any of these viruses, and other viral DNA segments thatfacilitate the uptake of the desired DNA segment by, and release into,the cytoplasm of cells and mixtures thereof. All of the above virusesmay require modification to render them non-pathogenic or lessantigenic. Other known vector systems, however, are also useful.

The present invention also relates to a method of treating a growthretardation disorder in a human subject, especially in a child oradolescent. The method involves genetically modifying a GH-responsive orgp130-responsive cell of a human subject having a growth retardationdisorder, typically resulting in short stature, such as, but not limitedto, dwarfism, GH deficiency, gonadal dysgenesis, chondrodystrophy,bone-cartilage dysplasia, or an idiopathic condition of severe shortstature. Typically, the cell is a pituitary, adrenal, hypothalamic,liver, immune-competent, or bone-forming cell that is responsive to agrowth-inducing cytokine in a paracrine manner. Examples includehepatocyte, lymphocyte, lymphocyte, chondrocyte, corticotroph,somatotroph, lactotroph, or gonadotroph cells, or cells derived from apituitary tumor, adrenal tumor, hypothalamic tumor, liver tumor, or bonetumor.

The cell(s) are genetically modified by any suitable method, in vivo orin vitro, for example by transfection or transduction, using a nucleicacid construct of the present invention, comprising a SOCS-3 promotersequence, or operative fragment thereof, operatively linked, in atranscriptional unit, to a DNA sequence encoding an RNA thatspecifically hybridizes to a functional SOCS-3 mRNA, i.e., aSOCS-3-specific antisense RNA. In response to the presence of agrowth-inducing cytokine, in vivo, the cell transcribes, from thetranscriptional unit, RNA transcript that hybridizes to SOCS-3 mRNA,preventing translation therefrom. This RNA transcript can be anantisense RNA or a catalytic RNA (ribozyme) that cleave the SOCS-3 mRNA.As a consequence, the amount of SOCS-3 protein produced within thegenetically modified cell(s) is reduced relative to unmodified cells ofthe same kind, and one or more symptoms of the growth retardationdisorder in the human subject are thereby improved, due to a lesseningof SOCS-3-mediated suppression of gp130-mediated signal transductionfrom growth-inducing cytokines, such as GH, within the geneticallymodified cell(s).

The present invention also relates to a method of treating a growthacceleration disorder in a human subject. The method involvesgenetically modifying a GH-responsive or gp130-responsive cell from atissue of a human subject having a growth acceleration disorder,resulting in greater than normal enlargement of one or more parts of thebody, such as, but not limited to, gigantism, acromegaly, Cushing'sdisease, or an idiopathic condition resulting in abnormal, non-edemicenlargement of bones, or facial or other soft tissue features.Typically, the cell is a pituitary, adrenal, hypothalamic, liver,immune-competent or bone-forming cell that is responsive to agrowth-inducing cytokine in a paracrine manner. Examples includehepatocyte, lymphocyte, chondrocyte, corticotroph, somatotroph,lactotroph, or gonadotroph cells, or cells derived from a pituitarytumor, adrenal tumor, hypothalamic tumor, liver tumor, or bone tumor.

The cell(s) are genetically modified by any suitable method, in vivo orin vitro, for example by transfection or transduction, using a nucleicacid construct, in accordance with the present invention, comprising aSOCS-3 promoter sequence, or operative fragment thereof, operativelylinked, in a transcriptional unit, to a DNA sequence encoding a SOCS-3protein, or functional fragment thereof. In response to agrowth-inducing hormone or cytokine, in vivo, SOCS-3 mRNA transcript istranscribed from the transcriptional unit, resulting in translation ofSOCS-3 message to SOCS-3 protein. The amount of SOCS-3 protein producedis thereby enhanced in the genetically modified cell(s) in response tothe presence of a growth-inducing cytokine, such as GH or a cytokine ofthe gp130 signaling subunit family, compared to the amount in unmodifiedcells of the same kind. The symptom(s) of the growth accelerationdisorder in the subject are thereby improved, due to increasedSOCS-3-mediated cytokine signal suppression within the geneticallymodified cell(s). Thus, for example, in pituitary corticotroph cells,ACTH secretion is suppressed by increased levels of SOCS-3, ultimatelyleading to less production of glucocorticoid hormones by the adrenalsand ameliorating symptoms of Cushing's disease. Similarly, the effectsof excess GH, as for example in acromegaly, are moderated in accordancewith the present method.

The present invention also relates to a method of treating an autoimmunedisease, immune disease, or inflammatory condition in a human subject.Such diseases or conditions include, but are not limited to, Crohn'sdisease, ulcerative colitis, multiple sclerosis, systemic lupuserythematosus, rheumatoid arthritis, Grave's disease, allergic oranaphylactic reactions, or neuroendocrinological responses topsychological or physical stress. The method involves geneticallymodifying a cell(s) from the subject that is gp130-responsive, i.e.,responsive to at least one pro-inflammatory cytokine, such as IL-6, LIF,or any other pro-inflammatory cytokine for which signal transduction isgp130-mediated. Typically, the cell is a pituitary, adrenal,hypothalamic, liver, intestinal, nerve, kidney, immune-competent, orbone-forming cell that is responsive to a pro-inflammatory cytokine in aparacrine manner. Examples include hepatocyte, lymphocyte, chondrocyte,neuron, intestinal epithelial, corticotroph, somatotroph, lactotroph, orgonadotroph cells.

The cell(s) are genetically modified by any suitable method, in vivo orin vitro, for example by transfection or transduction, using a nucleicacid construct comprising a SOCS-3 promoter sequence, or an operativefragment thereof, operatively linked, in a transcriptional unit, to aDNA sequence encoding a SOCS-3 protein, or functional fragment thereof.In response to an inflammatory cytokine of the gp130 signaling subunitfamily, in vivo, SOCS-3 mRNA transcript is transcribed from thetranscriptional unit, resulting in translation of SOCS-3 message toSOCS-3 protein. The amount of SOCS-3 protein produced is therebyenhanced in the genetically modified cell(s) in response to the presenceof an inflammatory cytokine of the gp130 signaling subunit family,compared to the amount in unmodified cells of the same kind. One or moresymptoms of the autoimmune disease, immune disease, or inflammatorycondition in the subject are thereby improved, due to a relativeincrease in SOCS-3-mediated signal suppression.

In another embodiment, the nucleic acid construct that is used in themethod comprises a SOCS-3 promoter sequence, or operative fragmentthereof, operatively linked, in a transcriptional unit, to a DNAsequence encoding a functional anti-inflammatory cytokine of the gp130signaling subunit cytokine family, such as IL-11, linked to a functionalsecretory signal. In response to the presence of a pro-inflammatorycytokine of the gp130 signaling subunit cytokine family, in vivo, theanti-inflammatory cytokine is produced and secreted by the modifiedcell(s), which has both paracrine and autocrine effects that improve Oneor more symptoms of the autoimmune disease, immune disease, orinflammatory condition in the subject.

In some embodiments of the present methods, gene delivery is done invitro, and the cell(s) is first obtained from a tissue of the humansubject by any suitable biopsy method, for example percutaneous biopsy,laparoscopic biopsy, or stereotactic cranial biopsy. Gene delivery isaccomplished in vitro, and the genetically modified cell(s) are thenre-implanted within the tissue of the human subject.

The nucleic acid construct that is used in the present methodsoptionally contains a reporter gene for convenient detection, isolationor selection of transgenic cells expressing from the SOCS-3 promoter asdescribed herein. For particular applications, other DNA sequencesencoding other preselected proteins are optionally linked to the SOCS-3promoter, making their expression inducible by LL-6-type cytokines andgp130-mediated signal transduction.

The present invention also relates to a kit for genetically modifying avertebrate cell. The kit is a ready assemblage of materials orcomponents for facilitating the genetic modification of a vertebratecell. The kit includes a polynucleotide comprising a murine SOCS-3promoter sequence having SEQ. ID. NO.:1, or an operative fragment ornon-murine homologue thereof, or an operative derivative of any ofthese, as described herein with respect to the nucleic acid construct ofthe present invention. Preferably the polynucleotide includes atranscriptional unit that contains the SOCS-3 promoter, operativelylinked to at least one DNA sequence encoding a preselected protein or toa gene-specific part thereof, such as a SOCS-3 protein, or a functionalfragment thereof, and/or a reporter gene for facilitating detection,isolation, or selection of genetically modified cells from unmodifiedcells. The DNA sequence encoding the preselected protein can be in asense or antisense orientation as appropriate for a particularapplication. Some embodiments of the kit are configured for use inpracticing the present methods of treating a growth retardation oracceleration disorder in a human subject or the present method oftreating an autoimmune disease, immune disease, or inflammatorycondition in a human subject.

The kit optionally contains a suitable transfecting agent, as describedabove. The kit includes instructions for using the materials orcomponents effectively. The materials or components assembled in the kitare provided to the practitioner stored in any convenient and suitableway that preserves their operability and utility. For example thecomponents can be in dissolved, dehydrated, or lyophilized form; theycan be provided at room, refrigerated or frozen temperatures.

The foregoing descriptions of the nucleic acid constructs, transgeniccells, transgenic vertebrates, methods, and kits of the presentinvention are illustrative and by no means exhaustive. The inventionwill now be described in greater detail by reference to the followingnon-limiting examples.

EXAMPLES Example 1

Materials and Methods

Materials. Recombinant murine LIF, IL-6, and IL-11 were purchased fromR&D Systems (Minneapolis, Minn.). Mouse liver Marathon-Ready® cDNA,Advantage®-GC cDNA polymerase, mouse GenomeWalker® Kit, andAdvantage®-GC genomic polymerase were from Clontech (Palo Alto, Calif.).Maxiscript® T7 polymerase kit and ribonuclease protection kit RPA-II®were from Ambion (Austin, Tex.). Polyclonal STAT-1 p84/p91 (M-22) andSTAT-3 (H-190) antibodies were from Santa Cruz Biotechnology (SantaCruz, Calif.). Mouse genomic DNA, Erase-a Base® system, pGL3 Basic andpSV-β-galactosidase vector were from Promega (Madison, Wis.). TOPO-TA®PCR2.1 vector was from Invitrogen (Carlsbad, Calif.).

Cell Culture. Cell culture of AtT-20/D16v-F2 cells was performed asdescribed (C. J. Auernhammer et al. [1998b]; C. J. Auernhammer et al.[1998a]). Individual clones of AtT-20 cells, overexpressing SOCS-3(AtT-20S), mock-transfected (AtT-20M), wild type STAT-3 (AtT-20W) ordominant negative STAT-3 mutants (AtT-20 F and AtT-20D), were isolatedafter stable transfection. (C. J. Auernhammer et al. [1998b]; C.Bousquet & S. Melmed, J. Biol. Chem. 274:10723-30 [1998]). From eachgroup, three separate individual clones with high stable overexpressionof the respective construct were selected with G418 (1 mg/mL) for theexperiments.

Northern blot analysis. Northern blot analysis was performed asdescribed (C. J. Auernhammer et al.[1998b]; C. J. Auernhammer et al.[1998a]). To detect endogenous SOCS-3 mRNA in AtT-20S cells, withouthybridization to exogenous SOCS-3 mRNA derived from stableoverexpression of SOCS-3, a probe spanning exon 1 and the untranslated5′ region of exon 2 as used. Otherwise, the previously described (C. J.Auernhammer et al. [1998b]; C. J. Auernhammer & S. Melmed, Endocrinology140:1559 [1999]) murine SOCS-3 probe spanning most of the coding regionof SOCS-3 was used.

5′-Rapid Amplification of cDNA Ends (RACE) and RNase protection Assay.5′-RACE was performed with a pre-made, adaptor-ligated Marathon-Ready®double stranded cDNA derived from pooled BALB/c mouse liver (A. Chenchiket al., Biotechnol. 3: 526-34 [1996]) and Advantage®-GC cDNA polymeraseusing gene-specific primary and nested antisense primers5′-CAGTAGAATCCGCTCTCCTGCAGCTTG-3′(SEQ.ID.NO.:15) and5′-CTCGCTTTTGGAGCTGAAGGTCTTGAG-3′ (SEQ. ID. NO.:16). Products werecloned into PCR2.1 vector, and multiple single clones sequenced.

RNase protection assay was performed with RPA-II® kit, following themanufacturer's recommendations. A fragment spanning nucleotides +158 to−275 was cloned into PCR.2.1 vector; the plasmid was linearized withBamHI, and a ³²P-UTP labeled antisense probe was generated with T7polymerase.

PCR-based characterization of the 5′-genomic region. The 5′-genomicregion of SOCS-3 was cloned using a PCR-based technique (P. D. Siebertet al., Nucleic Acids Res. 23:1087-88 [1995]) with pre-madeadaptor-ligated genomic DNA fragments, derived from ICR Swiss mice, asprovided by the Genomewalk® kit. PCR and subsequent nested PCR whereperformed by automatic hot-start as touchdown-PCR using Advantage®-GCgenomic Polymerase and gene specific antisense primers5′-CAGTAGAATCCGCTCTCCTGCAGCTTG-3′ (SEQ. ID. NO.:15) and5′-CTCGCTTTTGGAGCTGAAGGTCTTGAG-3′(SEQ.ID.NO.:16). Further genomic walksin the 5′ direction were performed with gene specific antisense primers5′-CTTCCTACCTAGTCCCGAAGCGAAATC-3′(SEQ.ID.NO.:17),5′-CAGATGTTGGCAGCCGTGAAGTCTAC-3′(SEQ.ID.NO.:18),5′-GCGGGCGAGTGTAGAGTCAGAGTTAGAG-3′(SEQ.ID.NO.:19), and5′-CGATTCCTGGAACTGCCCGGCCGGTCTTC-3′(SEQ.ID.NO.:20), as well as5′-CTCAGTGGGCTTTCTGACCTGCCCTCTTG-3′(SEQ.ID.NO.:21) and5′-GACTACACAGAGTAGCTTGGGCTAGGAG-3′(SEQ.ID.NO.:22). Products were clonedinto PCR2.1, and single clones were sequenced.

Different Constructs of the 5′ Genomic Region of SOCS-3. 3′-Truncatedforms of the full-length 3.7-kb construct in pGL3Basic vector (clone 6)were generated by PCR from genomic DNA and subsequent cloning asdescribed above.

5′-Truncated forms of clone 6 were generated using Erase-a-Base® kit,following the manufacturer's recommendations. Briefly, the 3.7-kbfull-length construct of the 5′ genomic region of SOCS-3 in pGL3Basicvector was digested with SstI and NheI, followed by unidirectionaldigestion with exonuclease III (S. Henikoff, Gene 28:351-59 [1984]) andsubsequent re-ligation.

Mutated forms of clone 6 were generated by overlap extension PCR (A.Aiyar et al., Methods Mol. Biol. 57:177-91 [1996]) with Pfu polymeraseand 5% DMSO, by using external sense primer 5′-CATCGCGACGCCCCCGCCTCT-3′(SEQ.ID.NO.:23) and antisense primer5′-GAAACCCGAGGGCCCCAGTCTG-3′(SEQ.ID.NO.:24) with exclusive restrictionsites for NruI or ApaI, respectively. Internal mutagenizing primerscaused deletions of nucleotides −80 to −60 and −101 to −62,respectively. Similarly, the STAT binding element region at −74 to −66was mutated. Gel-purified PCR-products and the original template weredigested with NruI and ApaI, fragments were purified, and the mutatedfragments were re-ligated into the original 3.7-kb construct inpGL3Basic vector. Each construct was verified by sequencing.

Luciferase Assay. For transient transfection experiments, 2×10⁵ cellswere plated in 6-well plates, incubated for 24 hours, and transfectedusing Lipofectamine-re and 0.5 μg of constructs in pGL3Basic vector, and1.0 μg pSV-β-galactosidase. Transfected cells were first incubated for24 hours in serum-free DMEM, followed by 6 hours of cytokine treatmentand subsequent measurement of luciferase activity. In experimentscomparing overexpressing dominant negative STAT-3 mutants or wild typeSOCS-3, treatment with LIF was for 45 minutes.

In experiments using different promoter constructs, transfectionefficiency was verified by measurement of β-galactosidase activity.

Electromobility shift assay. Nuclear extracts of AtT-20 cells andelectromobility shift assay (EMSA) were performed as described (P. D.Siebert et al., Nucleic Acids Res. 23:1087-88 [1995]). Briefly, AtT-20cells were grown to 80% confluence and were serum-deprived for 24 hoursbefore treatment with 10⁻⁹ M LIF, followed by cell lysis and preparationof nuclear extracts. For the EMSA, 20-μg nuclear extracts werepreincubated for 15 minutes at room temperature in 20 μL binding, buffer(10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% NP-40, 5%glycerol, 1 mg/mL BSA, pH 7.5) with 1 μg of poly(dI-dC). A ³²P-labeleddouble stranded oligonucleotide, corresponding to nucleotide sequence−77 to −57 of the SOCS-3 promoter(5′-⁷⁷CAGTTCCAGGAATCGGGGGGC⁻⁵⁷-3)(SEQ.ID.NO.:25), was used as a probe(60,000 cpm, 5 fmol per reaction) and added to each sample and bindingreaction, performed at room temperature for 20 min. In competitionexperiments, 100-fold molar excess of unlabeled double strandedcompetitor oligonucleotides were added to the preincubation reactionwith the double stranded oligonucleotide corresponding to nucleotidesequence −77 to −57 of the SOCS-3 promoter, this same oligonucleotidemutated at positions −74, −71, −69, and −66 (underlined)(5′-⁷⁷CAGATCGACGATTCGGGGGGC⁻⁵⁷-3)(SEQ.ID.NO.:26), or the AP-2recognition site oligonucleotide 5′-GATCGAACTGACCGCCCGCCGCCCGT-3′ (SEQ.ID. NO.:27). For supershift experiments 2 μg polyclonal STAT-1 p84/p91or STAT-3 antibody was added to the preincubation reaction and incubatedfor an additional 60 min at 4° C. Protein-DNA complexes were run on a 6%non-denaturing polyacrylamide gel in 0.5× TBE buffer (90 mM Tris, 64.6mM boric acid, 2.5 mM EDTA); gels were dried and autoradiographs wereexposed (Kodak biomax MS film at −70° C.).

Statistical analysis. Statistical analysis was performed by unpairedt-test. All values are mean±SEM.

Example 2

5′-Genomic Sequence of Murine SOCS-3 and Determination of theTranscription Start Site by 5′-RACE and RNase Protection Assay

Based on the sequence information from the 5′ genome walk, a full length5′ product of murine SOCS-3 spanning ˜3.8-kb of genomic sequence wasgenerated from ICR Swiss mice genomic DNA by PCR with Advantage®-GCgenomic polymerase using the following sense and antisense primers:

5′-GACGTTCCTAAAAGCATGCATGTCACC CAG-3′ (SEQ.ID.NO.:28) and

5′-GGATCTGCGCGGCGGTGGCTGCAGCTGCTT-3′ (SEQ.ID.NO.:29). Cloning of theproduct into PCR2.1 vector was followed by verification of senseorientation, sequencing, restriction enzyme digestion with SstI andXhoI, and subcloning of a ˜3.7-kb construct into pGL3Basic vector (clone6). Sequence information was obtained for the whole 3.8 kb. (SEQ. ID.NO.:1).

5′-RACE revealed the existence of an untranslated exon 1 (+1 to +289,SEQ. ID. NO.:32), separated from exon 2 (starting at +854; partial exon2, sequence +854 to +1033 [SEQ. ID. NO.:34]) by an intron (+290 to +853;SEQ. ID. NO.:33). Using Rnase protection assay, the main transcriptionstart site was defined and is referred to as +1. The previouslydetermined translation initiation site for murine SOCS-3 (GenBankAccession U88328) (R. Starr et al., Nature 387:917-21 [1997]) was inexon 2 at +944.

Example 3

Effects of Different Cytokines on SOCS-3 Promoter Activity and GeneExpression

FIG. 1 shows the stimulatory effect of various cytokines on expressionfrom the SOCS-3 promoter sequence. AtT-20 cells were either untreated,or stimulated with 0.5×10⁻⁹ M LIF, IL-6, or IL-11 for 60 or 120 min.Northern blot analysis showed a SOCS-3-specific signal of uniformtranscript size of ˜2.8 kb (FIG. 1A).

LIF was the most potent inducer of SOCS-3 mRNA expression. Although IL-6and IL-11 were less potent stimuli of SOCS-3 gene expression, they eachshowed a similar pattern of SOCS-3 mRNA induction. (FIG. 1A).

For measurement of SOCS-3 promoter activity, transient transfections ofAtT-20 cells were performed either with pGLBasic alone or with clone 6,a construct containing nucleotides −2,759 to +927 of the 5′-genomicregion of murine SOCS-3 linked to the luciferase reporter gene inpGL3Basic vector. (FIG. 1B). Relative SOCS-3 promoter activity isindicated by relative light unit values in FIG. 1B, calculated from 4independently performed experiments. Each experiment was performed withn=3 wells per group. Asterisks indicate in-group significance ofuntreated (−) vs. treated (+); *, P<0.05: **, P<0.01. AtT-20 cellstransfected with clone 6 showed a significantly higher basal luciferaseactivity than cells transfected with pGL3Basic alone (4043±443 vs.1611±398 relative light units [RLU]; P<0.001). Stimulation with 0.5×10⁻⁹M LIF, IL-6, or IL-11 caused no further increase of luciferase activityin control AtT-20 cells transfected with pGL3Basic alone. However, incomparison to untreated cells, AtT-20 cells transfected with clone 6showed an approximately 10-fold (P<0.0 1) increase in luciferaseactivity following stimulation with LIF, a 2-fold (not significant)increase following stimulation with IL-6, and a 3-fold (P<0.05)stimulation of luciferase activity by IL-11. (FIG. 1B).

Activation by LIF, IL-6 and IL-11, of SOCS-3 promoter activity and geneexpression thus is concordant with our finding of a functionallyimportant STAT-1/STAT-3 binding element in the murine SOCS-3 promoterregion.

Example 4

Effect of Overexpressed Dominant Negative STAT-3 Mutants or Wild TypeSOCS-3 on LIF-induced SOCS-3 Gene Expression and Promoter Activity

Expression from the SOCS-3 promoter is partly dependent both on theexpression of STAT-3 and SOCS-3 itself. FIG. 2 shows the effect ofoverexpressed dominant negative STAT-3 mutant or wild type SOCS-3 onLIF-induced SOCS-3 gene expression and promoter activity in AtT-20cells. AtT-20 cells overexpressing wild type STAT-3 (AtT-20W) showed a5.4±0.7-fold increase of SOCS-3 mRNA levels after stimulation with0.5×10⁻⁹ M LIF for 45 min. In comparison, AtT-20 cells overexpressingthe dominant negative mutants STAT-3F (AtT-20F) and STAT-3D (AtT-20D)both showed relatively diminished induction of SOCS-3 mRNA afterstimulation with LIF: 3.4±0.4 (p=0.07) and 2.6±0.1-fold (p<0.02),respectively. (FIGS. 2A, 2B). Similarly, transient transfectionexperiments with clone 6 showed stimulation of luciferase activity byLIF (6.9±0.5-fold) in AtT-20W cells, but only 5.4±0.5-(P=0.09) and3.4±0.4-fold (P<0.01) stimulation were observed in AtT-20F and AtT-20Dcells, respectively. (FIG. 2C). These results, showing that LIF-inducedSOCS-3 promoter activity and gene expression is decreased in thesedominant negative STAT-3 mutant transfectants, indicate that SOCS-3promoter activity is at least partly dependent on wild type STAT-3expression. (FIGS. 2A-C).

Overexpression of wild type SOCS-3 in AtT-20 cells abrogated LIF-inducedSOCS-3 promoter activity and gene expression. (FIGS. 2D-F).Mock-transfected AtT-20 cells (AtT-20M) showed an approximately 5-foldincrease of SOCS-3 mRNA levels after 45 min stimulation with 0.5×10⁻⁹ MLIF, while AtT-20 cells overexpressing wild type SOCS-3 (AtT-20S),showed a significant inhibition of LIF-induced SOCS-3 mRNA expression.(FIGS. 2D, 2E). Similarly, transient transfection experiments with clone6 revealed luciferase activity to be stimulated by LIF (9.9±1.3-fold) inAtT-20M cells, while LIF-induced luciferase activity in AtT-20S cellswas abrogated and did not differ substantially from luciferase activityin untreated AtT-20S cells. (FIG. 2F). These results indicate a negativeautoregulatory feedback of SOCS-3 on its own cytokine-induced geneexpression.

LIF-induced SOCS-3 mRNA and luciferase activity were each calculatedfrom 3 independently performed experiments. Each experiment wasperformed with 3 different clones per group. LIF-induced luciferaseactivity was normalized to the untreated control for each clone.

Example 5

Functional Analysis of Different SOCS-3 5′ Region-luciferase Constructs

Clone 6 (SEQ.ID.NO.:3) is the −2759 to +927 5′ genomic region of murineSOCS-3 linked to the luciferase reporter in pGL3Basic vector.3′-Truncations of clone 6 were: clone 4 (nt. −2759 to +104;SEQ.ID.NO.:2) and clone 2 (nt. −2759 to −716; SEQ.ID.NO.:38).5′-Truncations of clone 6 were: clone 6T1 (nt. −1864 to +927;SEQ.ID.NO.:4); clone 6T2 (nt. −857 to +927; SEQ.ID.NO.:5); clone 6T3(nt. −152 to +927; SEQ.ID.NO.: 12); and clone 6T4 (nt. −63 to +927;SEQ.ID.NO.:6). Analysis of clone 6 sequence with Mat Inspector V2.2 (K.Quandt et al., Nucleic Acids Res. 23: 4878-84 [1995]), revealedpotential STAT binding sites containing the consensus binding sequenceTT(N)₅AA (SEQ. ID. NO.:31) (C. M. Horvath et al., Genes Dev. 9:984-94[1995]; J. E. Darnell, Jr., Science 277:1630-35 [1997]; S. Becker etal., Nature 394:145-51 [1998]) located at nt. −97 to −89 and nt. −74 to−66, as well as at nt. −347 to −339 and −1403 to −1395. However, onlythe STAT binding site from nt. −74 to −66 showed the more specificsequence TTCCAGGAA (SEQ.ID.NO.:13), indicating a potential binding sitefor STAT-1 and STAT-3.

Therefore, in subsequent experiments, the focus centered on the STATbinding site at nt. −74 to −66, constituting part of the tandem STATbinding region pair of nt. −97 to −89 and nt. −74 to −66. Using overlapextension PCR, we deleted the complete tandem STAT binding region fromnt. −101 to −62 (clone 6D2; SEQ.ID.NO.:36), or only the 3′-located STATbinding element from nt. −80 to −60 (clone 6D1; SEQ.ID.NO.:35). In clone6M1 (SEQ.ID.NO.:37), the 3′-located STAT binding element from nt. −74 to−66 was not deleted, but mutated to ATCGACGAT, thus destroying thespecific binding sequence TTCCAGGAA (SEQ.ID.NO.:13). Clone 8(SEQ.ID.NO.:39) was a minimal −275 to +158 5′ genomic region of SOCS-3linked to the luciferase reporter in pGL3Basic vector. Basal andLIF-induced luciferase activity were assayed after transienttransfection of corticotroph AtT-20 cells with the different constructs.

FIG. 3 shows relative luciferase activities in transfected AtT-20 cellsbearing the different constructs. Relative luciferase activities werecalculated in comparison to basal luciferase activity of pGL3Basic alonewithout LIF treatment, which was defined as 1.0. Basal luciferaseactivity of clone 2 (SEQ.ID.NO.:38) did not differ from pGL3Basic, andneither clone 2 (SEQ.ID.NO.:38) nor pGL3Basic showed induction ofluciferase activity by LIF. However, clones 4 and 6 (SEQ.ID.NOS.:2 and3, respectively) showed 7- and 4-fold higher basal luciferase activity,respectively, as well as 35-fold higher LIF-stimulated luciferaseactivity, compared to the pGL3Basic (P<0.01). This indicates that theregion from nt. +104 to +927 is not involved in SOCS-3 promoteractivity.

Increasing 5′-truncations of clone 6 up to nt. −161 caused a gradualincrease of basal and LIF-stimulated luciferase activity, with bothclone 6T2 (P<0.01) and clone 6T3 (P<0.001) showing significantly higherbasal and LIF-induced luciferase activities than clone 6. Clone 6T3 hadthe highest basal (17-fold elevation) and LIF-induced (97-foldelevation) luciferase activities, compared to basal pGL3Basic (p<0.001).This demonstrates that the region from nt. −2759 to −161 containsapparent negative regulator elements, but is not responsible for basaland LIF-induced SOCS-3 promoter activity.

Further 5′-truncation to nt. −63 in clone 6T4 (SEQ.ID.NO.:6) causeddecreases in basal activity and, more markedly, in LIF-induciblepromoter activity. Mutated clones 6D1 (SEQ.ID.NO.:35) (P<0.001) and 6M1(SEQ.ID.NO.:37) (P<0.01) showed reduced LIF-induced luciferase activity,compared to wild type clone 6 (SEQ.ID.NO.:3). (FIG. 3). Extending thedeletion to the entire tandem STAT binding region in clone 6D2(SEQ.ID.NO.:36), showed no significant difference in the magnitude ofbasal vs. LIF-induced luciferase activity in comparison to clone 6D1(SEQ.ID.NO.:35). These results indicate that the specific STAT-1/STAT-3binding element at −74 to −66 (TTCCAGGAA; SEQ.ID.NO.:13) mediates theLIF-induced rise in luciferase activity, while the more 5′-located STATbinding element at −97 to −89 (TTACAAGAA; SEQ.ID.NO.:30) does notsignificantly participate in this signal.

Clone 8 (SEQ.ID.NO.:39) showed basal and LIF-induced luciferase activitycomparable to clone 6 (SEQ.ID.NO.:3). (FIG. 3). This furtherdemonstrates the functional importance for SOCS-3 promoter activity ofthe region containing the STAT-1/STAT-3 binding element.

Example 6

Electromobility Shift Assay

EMSA showed specific binding of nuclear extracts from LIF-induced AtT-20cells to a double stranded oligonucleotide probe spanning nt. −77 to −57(STAT oligoprobe), including the STAT-1/STAT-3 binding element from −74to −66. While nuclear extracts from unstimulated AtT-20 cells did notform specific complexes with the oligoprobe, nuclear extracts fromLIF-stimulated AtT-20 cells formed three specific complexes, compatiblewith STAT-3 homodimers, STAT-1/STAT-3 heterodimers and STAT-1 homodimers(C. M. Horvath et al., Genes Dev. 9:984-94 [1995]; J. E. Darnell, Jr.,Science 277:1630-35 [1997]; S. Becker et al., Nature 394:145-51 [1998]).The three complexes disappeared during self-competition with a 100-foldexcess of unlabeled double stranded STAT oligonucleotide, whereas thesame double stranded oligonucleotide mutated at positions −74, −71, −69,and −66, or a nonspecific double stranded AP-2 oligonucleotide had noeffect. Incubation with a specific antibody directed against STAT-1abolished the two bands representing STAT-1 homodimer and STAT1/STAT3heterodimer. Similarly, incubation with a specific antibody directedagainst STAT-3 abolished the two bands representing STAT-3 homodimer andSTAT1/STAT3 heterodimer. These results are evidence of specific bindingof STAT-1 and STAT-3 to the SOCS-3 promoter region between nt. −74 to−66.

The foregoing examples being illustrative but not an exhaustivedescription of the embodiments of the present invention, the followingclaims are presented.

39 1 3940 DNA Mus musculus promoter (-2907)...(1033) promoter (0)...(0)promoter (0)...(0) promoter (0)...(0) promoter (0)...(0) promoter(0)...(0) promoter (0)...(0) 1 gacgttccta aaagcatgca tgtcacccagcttacccacc catctcaggc cacagcagcc 60 tgagagagcg gaagaacacc tgctggtcctgtcccacctc tcctcttcaa acagccccac 120 atcctccagt tttgctctgg gtggagctccctgctggccc tgcagaggga aggctctcct 180 aagcatcatc tatcagaacg tcttcaaaaaaaaaaaaaaa aaaaaaaaag cctctccagc 240 caggctagct ctaacaccat ttcttccccttcccctctct caaattcact tatctttttt 300 tttttttttt tttttggatt tttgagacagggtttctctg tatagccctg gttgtcctgg 360 aactcacttt gtacaccagc ctggcctcgaactcagagat ccacctgcct ctgcctcctg 420 agtgctggga ttaaaggcgt gcgccaccacgcccggctaa attcacttat ctatttaatg 480 tatatagggt ataggctgcc cttgaactcacaaagatctg cttgctttgc ttctggaata 540 ctaaaggtgt gtgctaccat cacagggaccaagatttatt ttaattctgt atatgtgtgt 600 gtgtgtgtgt gtgtatgggg ggtgcacatgagtacagatt cccttggagg cctggggtgg 660 cttaggactg gggttacaac agttgtgacccatcctacat aggtcctggc accaacaccc 720 cccccccccc cccccgtctt ccagaagtgcagcaggtgtt cttaactgct gagccagcaa 780 tccagcccct gacttccctc tcttacttaagaagctatca cagtgtctca ctgggtcaca 840 atcatgacta gtccttgctc atggcccacagcctcttccc cactgtgggt tttgccccgc 900 agctctgccg ccccagcgct gcacccgaggcctgacagag ccaggcacga agtcagggtt 960 tgtggaatgg atgaatgaac ttgactcgtggcagagcatt gtaatttaca aagcactttc 1020 ccatccatta actccagggc tatttcctaagagtcctccc tgtcctccac tgccctcggc 1080 tcagaggcat acggtcaagg cagtggctggggaacactcc ctgaatgaga tcaaggaggg 1140 cttgttcaca gagaaaggga gaatccatttggggagcctg agagtgactc gaaggcaagg 1200 actgggcctc acctgtggga tctccatctgtgagcatccg ctcatcagac cagtgtgaga 1260 tattttaaat aaggccccta agcctcttgactactggaat tgccaggggc gggggacaga 1320 tgggcaccca tccctattta acagataacaagactgagtc cagagaggca gtgcacctgc 1380 cctggtctct cttagttcct cagcatcagtggagcagatt ggacacagtg ggccagaagg 1440 gaagcaggca gccctccctc ctagcccaagctactctgtg tagtcagttt gccctcctac 1500 tggtgttaca agaagcctgt ggtatccaagagggcaggtc agaaagccca ctgagagcag 1560 acactgtgtg tcacttagct ggttctcaggtggctgccac ttcctgctgc ctgttgcaaa 1620 actcgacact aggcctttat agatactcacgtgaccagga gtaaacaacc tttcacctca 1680 atcacctgct cttatcaata ctccctctccaccccaccat cgggaaagtt cagacacctt 1740 aaaacgtaga ggcaagagag ggtccattctgacacctcag cgactttcag gcagtggctg 1800 aacccgttac aacgctctgt ggacagtcctcctagtcgac attccttctc aggtttgacc 1860 ctgtcctggg aagtgaggct tctctctctgggttccccac tcctgttctt gaataaggag 1920 ccccacaacc tcttattctc tctatacagagcctgggaaa cagcaaaact cggctcgcct 1980 acaagactcc agcgcgccct ctggtggactcgggggacga gcatgggatg agggtttctt 2040 tcctctagct cccccaccgc gccgagagtacctgggcgga cccacagttc gccacgcagg 2100 ttgggaggcc cagatgagtg ataaggtagtagttagctgc tcctcccacc ccactcccca 2160 aaggacatca gcacccacgt ctgtcaccgaagaaccaggc aatgggcgga tgagctgagg 2220 ccaggtagct gcttctaagt cagtgtctcctccacttctg gatctcacag cttcatcttt 2280 tggacctgtc tacaggtaaa tgtcgcgcatccccctcctc cacttcctag gtccccagtg 2340 ggctggtggc tgaatggtcc tacgtcccttttggttggca cgggatgctt ggaactgtac 2400 atgaggacct cggggtggcc tgggtgcagagggaggggag cgtccccgcg gggatcaaaa 2460 gaaagggaag gggtgccagg agggagcctctcccggctgg cctcctagaa ctgcccgcgc 2520 gctcccatcg cgacgccccc gcctctgccagaaaccagcc ttcttagaag ggaggggggg 2580 gaaagtgtga atgagaagtt gggggcggagcgcgcggggg aggggccgct gccaggaacg 2640 ctcggccaag gctggcgccg cgcccgccggtcgggcagcc tcgcgccgcg ctttgtctcc 2700 ctctcggtga gtctcggcgg gtcctggaggccccagctcc aagcccgccc tccgcagccc 2760 ctccctcgcc ctccgcgcac agcctttcagtgcagagtag tgactaaaca ttacaagaag 2820 accggccggg cagttccagg aatcggggggcggggcgtac tggccgggta aatacccgcg 2880 cgcgcggcct ccgaggcggc tctaactctgactctacact cgcccgctcc tacgaccgct 2940 gtctctccgg gctcccggac gcccccttcccggcccagct ctccgtcgag gtccctcgcc 3000 caggtccttt gcctgattcg cccaggagtgcgcctcatcg gcccggggag cagcgaagcc 3060 agagggggcg cacgcacggg gagcccctttgtagacttca cggctgccaa catctgggcg 3120 cagcgcgagc cactgctggg cgccgcctcgcctcggggac cataggaggc gcagccccaa 3180 ggccggagat ttcgcttcgg gactaggtaggaaggagggg cgcggtgtgg ggaagggtgg 3240 gggcatcggt ccagctcggg agcttttcccggtttctcct ccccttcccg ggtcattccc 3300 ggtagggagg ggacgaggca gggggcagagcggatgagaa ccgaagatcc ctgattcccg 3360 tcatactcag actggggccc tcgggtttctcctgtcccct ctctcacata tctcgggttt 3420 ggcacccccc ttttttcgcc ctcgccactgaggacaccgg actgagaggc gccctgagcg 3480 tccctagggc tcttgtgtct ctccccatcctggccgcgct cctggagacc caacttccac 3540 gcgcgagttt tctctgggcg tcctcctagggcgggcaggg gaagagactg tctggggttg 3600 gccggcagtg accgaggaca gtcgagttccgcgaggtggc tgggcctgag acacggtcta 3660 aagcggggca aaggggtgcc ccgggcgctaggcggaggct ggagggccgg gcacgctgga 3720 gggttccggg cactcacgcg cctcacgctttgctctctgc agctccccgg gatgcggtag 3780 cggccgctgt gcggaggccg cgaagcagctgcagccaccg ccgcgcagat ccacgctggc 3840 tccgtgcgcc atggtcaccc acagcaagtttcccgccgcc gggatgagcc gccccctgga 3900 caccagcctg cgcctcaaga ccttcagctccaaaagcgag 3940 2 2863 DNA Mus musculus promoter (-2759)...(104) 2ccctgctggc cctgcagagg gaaggctctc ctaagcatca tctatcagaa cgtcttcaaa 60aaaaaaaaaa aaaaaaaaaa agcctctcca gccaggctag ctctaacacc atttcttccc 120cttcccctct ctcaaattca cttatctttt tttttttttt tttttttgga tttttgagac 180agggtttctc tgtatagccc tggttgtcct ggaactcact ttgtacacca gcctggcctc 240gaactcagag atccacctgc ctctgcctcc tgagtgctgg gattaaaggc gtgcgccacc 300acgcccggct aaattcactt atctatttaa tgtatatagg gtataggctg cccttgaact 360cacaaagatc tgcttgcttt gcttctggaa tactaaaggt gtgtgctacc atcacaggga 420ccaagattta ttttaattct gtatatgtgt gtgtgtgtgt gtgtgtatgg ggggtgcaca 480tgagtacaga ttcccttgga ggcctggggt ggcttaggac tggggttaca acagttgtga 540cccatcctac ataggtcctg gcaccaacac cccccccccc cccccccgtc ttccagaagt 600gcagcaggtg ttcttaactg ctgagccagc aatccagccc ctgacttccc tctcttactt 660aagaagctat cacagtgtct cactgggtca caatcatgac tagtccttgc tcatggccca 720cagcctcttc cccactgtgg gttttgcccc gcagctctgc cgccccagcg ctgcacccga 780ggcctgacag agccaggcac gaagtcaggg tttgtggaat ggatgaatga acttgactcg 840tggcagagca ttgtaattta caaagcactt tcccatccat taactccagg gctatttcct 900aagagtcctc cctgtcctcc actgccctcg gctcagaggc atacggtcaa ggcagtggct 960ggggaacact ccctgaatga gatcaaggag ggcttgttca cagagaaagg gagaatccat 1020ttggggagcc tgagagtgac tcgaaggcaa ggactgggcc tcacctgtgg gatctccatc 1080tgtgagcatc cgctcatcag accagtgtga gatattttaa ataaggcccc taagcctctt 1140gactactgga attgccaggg gcgggggaca gatgggcacc catccctatt taacagataa 1200caagactgag tccagagagg cagtgcacct gccctggtct ctcttagttc ctcagcatca 1260gtggagcaga ttggacacag tgggccagaa gggaagcagg cagccctccc tcctagccca 1320agctactctg tgtagtcagt ttgccctcct actggtgtta caagaagcct gtggtatcca 1380agagggcagg tcagaaagcc cactgagagc agacactgtg tgtcacttag ctggttctca 1440ggtggctgcc acttcctgct gcctgttgca aaactcgaca ctaggccttt atagatactc 1500acgtgaccag gagtaaacaa cctttcacct caatcacctg ctcttatcaa tactccctct 1560ccaccccacc atcgggaaag ttcagacacc ttaaaacgta gaggcaagag agggtccatt 1620ctgacacctc agcgactttc aggcagtggc tgaacccgtt acaacgctct gtggacagtc 1680ctcctagtcg acattccttc tcaggtttga ccctgtcctg ggaagtgagg cttctctctc 1740tgggttcccc actcctgttc ttgaataagg agccccacaa cctcttattc tctctataca 1800gagcctggga aacagcaaaa ctcggctcgc ctacaagact ccagcgcgcc ctctggtgga 1860ctcgggggac gagcatggga tgagggtttc tttcctctag ctcccccacc gcgccgagag 1920tacctgggcg gacccacagt tcgccacgca ggttgggagg cccagatgag tgataaggta 1980gtagttagct gctcctccca ccccactccc caaaggacat cagcacccac gtctgtcacc 2040gaagaaccag gcaatgggcg gatgagctga ggccaggtag ctgcttctaa gtcagtgtct 2100cctccacttc tggatctcac agcttcatct tttggacctg tctacaggta aatgtcgcgc 2160atccccctcc tccacttcct aggtccccag tgggctggtg gctgaatggt cctacgtccc 2220ttttggttgg cacgggatgc ttggaactgt acatgaggac ctcggggtgg cctgggtgca 2280gagggagggg agcgtccccg cggggatcaa aagaaaggga aggggtgcca ggagggagcc 2340tctcccggct ggcctcctag aactgcccgc gcgctcccat cgcgacgccc ccgcctctgc 2400cagaaaccag ccttcttaga agggaggggg gggaaagtgt gaatgagaag ttgggggcgg 2460agcgcgcggg ggaggggccg ctgccaggaa cgctcggcca aggctggcgc cgcgcccgcc 2520ggtcgggcag cctcgcgccg cgctttgtct ccctctcggt gagtctcggc gggtcctgga 2580ggccccagct ccaagcccgc cctccgcagc ccctccctcg ccctccgcgc acagcctttc 2640agtgcagagt agtgactaaa cattacaaga agaccggccg ggcagttcca ggaatcgggg 2700ggcggggcgt actggccggg taaatacccg cgcgcgcggc ctccgaggcg gctctaactc 2760tgactctaca ctcgcccgct cctacgaccg ctgtctctcc gggctcccgg acgccccctt 2820cccggcccag ctctccgtcg aggtccctcg cccaggtcct ttg 2863 3 3686 DNA Musmusculus promoter (-2759)...(927) 3 ccctgctggc cctgcagagg gaaggctctcctaagcatca tctatcagaa cgtcttcaaa 60 aaaaaaaaaa aaaaaaaaaa agcctctccagccaggctag ctctaacacc atttcttccc 120 cttcccctct ctcaaattca cttatctttttttttttttt tttttttgga tttttgagac 180 agggtttctc tgtatagccc tggttgtcctggaactcact ttgtacacca gcctggcctc 240 gaactcagag atccacctgc ctctgcctcctgagtgctgg gattaaaggc gtgcgccacc 300 acgcccggct aaattcactt atctatttaatgtatatagg gtataggctg cccttgaact 360 cacaaagatc tgcttgcttt gcttctggaatactaaaggt gtgtgctacc atcacaggga 420 ccaagattta ttttaattct gtatatgtgtgtgtgtgtgt gtgtgtatgg ggggtgcaca 480 tgagtacaga ttcccttgga ggcctggggtggcttaggac tggggttaca acagttgtga 540 cccatcctac ataggtcctg gcaccaacaccccccccccc cccccccgtc ttccagaagt 600 gcagcaggtg ttcttaactg ctgagccagcaatccagccc ctgacttccc tctcttactt 660 aagaagctat cacagtgtct cactgggtcacaatcatgac tagtccttgc tcatggccca 720 cagcctcttc cccactgtgg gttttgccccgcagctctgc cgccccagcg ctgcacccga 780 ggcctgacag agccaggcac gaagtcagggtttgtggaat ggatgaatga acttgactcg 840 tggcagagca ttgtaattta caaagcactttcccatccat taactccagg gctatttcct 900 aagagtcctc cctgtcctcc actgccctcggctcagaggc atacggtcaa ggcagtggct 960 ggggaacact ccctgaatga gatcaaggagggcttgttca cagagaaagg gagaatccat 1020 ttggggagcc tgagagtgac tcgaaggcaaggactgggcc tcacctgtgg gatctccatc 1080 tgtgagcatc cgctcatcag accagtgtgagatattttaa ataaggcccc taagcctctt 1140 gactactgga attgccaggg gcgggggacagatgggcacc catccctatt taacagataa 1200 caagactgag tccagagagg cagtgcacctgccctggtct ctcttagttc ctcagcatca 1260 gtggagcaga ttggacacag tgggccagaagggaagcagg cagccctccc tcctagccca 1320 agctactctg tgtagtcagt ttgccctcctactggtgtta caagaagcct gtggtatcca 1380 agagggcagg tcagaaagcc cactgagagcagacactgtg tgtcacttag ctggttctca 1440 ggtggctgcc acttcctgct gcctgttgcaaaactcgaca ctaggccttt atagatactc 1500 acgtgaccag gagtaaacaa cctttcacctcaatcacctg ctcttatcaa tactccctct 1560 ccaccccacc atcgggaaag ttcagacaccttaaaacgta gaggcaagag agggtccatt 1620 ctgacacctc agcgactttc aggcagtggctgaacccgtt acaacgctct gtggacagtc 1680 ctcctagtcg acattccttc tcaggtttgaccctgtcctg ggaagtgagg cttctctctc 1740 tgggttcccc actcctgttc ttgaataaggagccccacaa cctcttattc tctctataca 1800 gagcctggga aacagcaaaa ctcggctcgcctacaagact ccagcgcgcc ctctggtgga 1860 ctcgggggac gagcatggga tgagggtttctttcctctag ctcccccacc gcgccgagag 1920 tacctgggcg gacccacagt tcgccacgcaggttgggagg cccagatgag tgataaggta 1980 gtagttagct gctcctccca ccccactccccaaaggacat cagcacccac gtctgtcacc 2040 gaagaaccag gcaatgggcg gatgagctgaggccaggtag ctgcttctaa gtcagtgtct 2100 cctccacttc tggatctcac agcttcatcttttggacctg tctacaggta aatgtcgcgc 2160 atccccctcc tccacttcct aggtccccagtgggctggtg gctgaatggt cctacgtccc 2220 ttttggttgg cacgggatgc ttggaactgtacatgaggac ctcggggtgg cctgggtgca 2280 gagggagggg agcgtccccg cggggatcaaaagaaaggga aggggtgcca ggagggagcc 2340 tctcccggct ggcctcctag aactgcccgcgcgctcccat cgcgacgccc ccgcctctgc 2400 cagaaaccag ccttcttaga agggaggggggggaaagtgt gaatgagaag ttgggggcgg 2460 agcgcgcggg ggaggggccg ctgccaggaacgctcggcca aggctggcgc cgcgcccgcc 2520 ggtcgggcag cctcgcgccg cgctttgtctccctctcggt gagtctcggc gggtcctgga 2580 ggccccagct ccaagcccgc cctccgcagcccctccctcg ccctccgcgc acagcctttc 2640 agtgcagagt agtgactaaa cattacaagaagaccggccg ggcagttcca ggaatcgggg 2700 ggcggggcgt actggccggg taaatacccgcgcgcgcggc ctccgaggcg gctctaactc 2760 tgactctaca ctcgcccgct cctacgaccgctgtctctcc gggctcccgg acgccccctt 2820 cccggcccag ctctccgtcg aggtccctcgcccaggtcct ttgcctgatt cgcccaggag 2880 tgcgcctcat cggcccgggg agcagcgaagccagaggggg cgcacgcacg gggagcccct 2940 ttgtagactt cacggctgcc aacatctgggcgcagcgcga gccactgctg ggcgccgcct 3000 cgcctcgggg accataggag gcgcagccccaaggccggag atttcgcttc gggactaggt 3060 aggaaggagg ggcgcggtgt ggggaagggtgggggcatcg gtccagctcg ggagcttttc 3120 ccggtttctc ctccccttcc cgggtcattcccggtaggga ggggacgagg cagggggcag 3180 agcggatgag aaccgaagat ccctgattcccgtcatactc agactggggc cctcgggttt 3240 ctcctgtccc ctctctcaca tatctcgggtttggcacccc ccttttttcg ccctcgccac 3300 tgaggacacc ggactgagag gcgccctgagcgtccctagg gctcttgtgt ctctccccat 3360 cctggccgcg ctcctggaga cccaacttccacgcgcgagt tttctctggg cgtcctccta 3420 gggcgggcag gggaagagac tgtctggggttggccggcag tgaccgagga cagtcgagtt 3480 ccgcgaggtg gctgggcctg agacacggtctaaagcgggg caaaggggtg ccccgggcgc 3540 taggcggagg ctggagggcc gggcacgctggagggttccg ggcactcacg cgcctcacgc 3600 tttgctctct gcagctcccc gggatgcggtagcggccgct gtgcggaggc cgcgaagcag 3660 ctgcagccac cgccgcgcag atccac 36864 2791 DNA Mus musculus promoter (-1864)...(927) 4 ttcctaagag tcctccctgtcctccactgc cctcggctca gaggcatacg gtcaaggcag 60 tggctgggga acactccctgaatgagatca aggagggctt gttcacagag aaagggagaa 120 tccatttggg gagcctgagagtgactcgaa ggcaaggact gggcctcacc tgtgggatct 180 ccatctgtga gcatccgctcatcagaccag tgtgagatat tttaaataag gcccctaagc 240 ctcttgacta ctggaattgccaggggcggg ggacagatgg gcacccatcc ctatttaaca 300 gataacaaga ctgagtccagagaggcagtg cacctgccct ggtctctctt agttcctcag 360 catcagtgga gcagattggacacagtgggc cagaagggaa gcaggcagcc ctccctccta 420 gcccaagcta ctctgtgtagtcagtttgcc ctcctactgg tgttacaaga agcctgtggt 480 atccaagagg gcaggtcagaaagcccactg agagcagaca ctgtgtgtca cttagctggt 540 tctcaggtgg ctgccacttcctgctgcctg ttgcaaaact cgacactagg cctttataga 600 tactcacgtg accaggagtaaacaaccttt cacctcaatc acctgctctt atcaatactc 660 cctctccacc ccaccatcgggaaagttcag acaccttaaa acgtagaggc aagagagggt 720 ccattctgac acctcagcgactttcaggca gtggctgaac ccgttacaac gctctgtgga 780 cagtcctcct agtcgacattccttctcagg tttgaccctg tcctgggaag tgaggcttct 840 ctctctgggt tccccactcctgttcttgaa taaggagccc cacaacctct tattctctct 900 atacagagcc tgggaaacagcaaaactcgg ctcgcctaca agactccagc gcgccctctg 960 gtggactcgg gggacgagcatgggatgagg gtttctttcc tctagctccc ccaccgcgcc 1020 gagagtacct gggcggacccacagttcgcc acgcaggttg ggaggcccag atgagtgata 1080 aggtagtagt tagctgctcctcccacccca ctccccaaag gacatcagca cccacgtctg 1140 tcaccgaaga accaggcaatgggcggatga gctgaggcca ggtagctgct tctaagtcag 1200 tgtctcctcc acttctggatctcacagctt catcttttgg acctgtctac aggtaaatgt 1260 cgcgcatccc cctcctccacttcctaggtc cccagtgggc tggtggctga atggtcctac 1320 gtcccttttg gttggcacgggatgcttgga actgtacatg aggacctcgg ggtggcctgg 1380 gtgcagaggg aggggagcgtccccgcgggg atcaaaagaa agggaagggg tgccaggagg 1440 gagcctctcc cggctggcctcctagaactg cccgcgcgct cccatcgcga cgcccccgcc 1500 tctgccagaa accagccttcttagaaggga ggggggggaa agtgtgaatg agaagttggg 1560 ggcggagcgc gcgggggaggggccgctgcc aggaacgctc ggccaaggct ggcgccgcgc 1620 ccgccggtcg ggcagcctcgcgccgcgctt tgtctccctc tcggtgagtc tcggcgggtc 1680 ctggaggccc cagctccaagcccgccctcc gcagcccctc cctcgccctc cgcgcacagc 1740 ctttcagtgc agagtagtgactaaacatta caagaagacc ggccgggcag ttccaggaat 1800 cggggggcgg ggcgtactggccgggtaaat acccgcgcgc gcggcctccg aggcggctct 1860 aactctgact ctacactcgcccgctcctac gaccgctgtc tctccgggct cccggacgcc 1920 cccttcccgg cccagctctccgtcgaggtc cctcgcccag gtcctttgcc tgattcgccc 1980 aggagtgcgc ctcatcggcccggggagcag cgaagccaga gggggcgcac gcacggggag 2040 cccctttgta gacttcacggctgccaacat ctgggcgcag cgcgagccac tgctgggcgc 2100 cgcctcgcct cggggaccataggaggcgca gccccaaggc cggagatttc gcttcgggac 2160 taggtaggaa ggaggggcgcggtgtgggga agggtggggg catcggtcca gctcgggagc 2220 ttttcccggt ttctcctccccttcccgggt cattcccggt agggagggga cgaggcaggg 2280 ggcagagcgg atgagaaccgaagatccctg attcccgtca tactcagact ggggccctcg 2340 ggtttctcct gtcccctctctcacatatct cgggtttggc accccccttt tttcgccctc 2400 gccactgagg acaccggactgagaggcgcc ctgagcgtcc ctagggctct tgtgtctctc 2460 cccatcctgg ccgcgctcctggagacccaa cttccacgcg cgagttttct ctgggcgtcc 2520 tcctagggcg ggcaggggaagagactgtct ggggttggcc ggcagtgacc gaggacagtc 2580 gagttccgcg aggtggctgggcctgagaca cggtctaaag cggggcaaag gggtgccccg 2640 ggcgctaggc ggaggctggagggccgggca cgctggaggg ttccgggcac tcacgcgcct 2700 cacgctttgc tctctgcagctccccgggat gcggtagcgg ccgctgtgcg gaggccgcga 2760 agcagctgca gccaccgccgcgcagatcca c 2791 5 1803 DNA Mus musculus promoter (-857)...(927) 5gggtttcttt cctctagctc ccccaccgcg ccgagagtac ctgggcggac ccacagttcg 60ccacgcaggt tgggaggccc agatgagtga taaggtagta gttagctgct cctcccaccc 120cactccccaa aggacatcag cacccacgtc tgtcaccgaa gaaccaggca atgggcggat 180gagctgaggc caggtagctg cttctaagtc agtgtctcct ccacttctgg atctcacagc 240ttcatctttt ggacctgtct acaggtaaat gtcgcgcatc cccctcctcc acttcctagg 300tccccagtgg gctggtggct gaatggtcct acgtcccttt tggttggcac gggatgcttg 360gaactgtaca tgaggacctc ggggtggcct gggtgcagag ggaggggagc gtccccgcgg 420ggatcaaaag aaagggaagg ggtgccagga gggagcctct cccggctggc ctcctagaac 480tgcccgcgcg ctcccatcgc gacgcccccg cctctgccag aaaccagcct tcttagaagg 540gagggggggg aaagtgtgaa tgagaagttg ggggcggagc gcgcggggga ggggccgctg 600ccaggaacgc tcggccaagg ctggcgccgc gcccgccggt cgggcagcct cgcgccgcgc 660tttgtctccc tctcggtgag tctcggcggg tcctggaggc cccagctcca agcccgccct 720ccgcagcccc tccctcgccc tccgcgcaca gcctttcagt gcagagtagt gactaaacat 780tacaagaaga ccggccgggc agttccagga atcggggggc ggggcgtact ggccgggtaa 840atacccgcgc gcgcggcctc cgaggcggct ctaactctga ctctacactc gcccgctcct 900acgaccgctg tctctccggg ctcccggacg cccccttccc ggcccagctc tccgtcgagg 960tccctcgccc aggtcctttg cctgattcgc ccaggagtgc gcctcatcgg cccggggagc 1020agcgaagcca gagggggcgc acgcacgggg agcccctttg tagacttcac ggctgccaac 1080atctgggcgc agcgcgagcc actgctgggc gccgcctcgc ctcggggacc ataggaggcg 1140cagccccaag gccggagatt tcgcttcggg actaggtagg aaggaggggc gcggtgtggg 1200gaagggtggg ggcatcggtc cagctcggga gcttttcccg gtttctcctc cccttcccgg 1260gtcattcccg gtagggaggg gacgaggcag ggggcagagc ggatgagaac cgaagatccc 1320tgattcccgt catactcaga ctggggccct cgggtttctc ctgtcccctc tctcacatat 1380ctcgggtttg gcacccccct tttttcgccc tcgccactga ggacaccgga ctgagaggcg 1440ccctgagcgt ccctagggct cttgtgtctc tccccatcct ggccgcgctc ctggagaccc 1500aacttccacg cgcgagtttt ctctgggcgt cctcctaggg cgggcagggg aagagactgt 1560ctggggttgg ccggcagtga ccgaggacag tcgagttccg cgaggtggct gggcctgaga 1620cacggtctaa agcggggcaa aggggtgccc cgggcgctag gcggaggctg gagggccggg 1680cacgctggag ggttccgggc actcacgcgc ctcacgcttt gctctctgca gctccccggg 1740atgcggtagc ggccgctgtg cggaggccgc gaagcagctg cagccaccgc cgcgcagatc 1800cac 1803 6 990 DNA Mus musculus promoter (-63)...(927) 6 ggggggcggggcgtactggc cgggtaaata cccgcgcgcg cggcctccga ggcggctcta 60 actctgactctacactcgcc cgctcctacg accgctgtct ctccgggctc ccggacgccc 120 ccttcccggcccagctctcc gtcgaggtcc ctcgcccagg tcctttgcct gattcgccca 180 ggagtgcgcctcatcggccc ggggagcagc gaagccagag ggggcgcacg cacggggagc 240 ccctttgtagacttcacggc tgccaacatc tgggcgcagc gcgagccact gctgggcgcc 300 gcctcgcctcggggaccata ggaggcgcag ccccaaggcc ggagatttcg cttcgggact 360 aggtaggaaggaggggcgcg gtgtggggaa gggtgggggc atcggtccag ctcgggagct 420 tttcccggtttctcctcccc ttcccgggtc attcccggta gggaggggac gaggcagggg 480 gcagagcggatgagaaccga agatccctga ttcccgtcat actcagactg gggccctcgg 540 gtttctcctgtcccctctct cacatatctc gggtttggca cccccctttt ttcgccctcg 600 ccactgaggacaccggactg agaggcgccc tgagcgtccc tagggctctt gtgtctctcc 660 ccatcctggccgcgctcctg gagacccaac ttccacgcgc gagttttctc tgggcgtcct 720 cctagggcgggcaggggaag agactgtctg gggttggccg gcagtgaccg aggacagtcg 780 agttccgcgaggtggctggg cctgagacac ggtctaaagc ggggcaaagg ggtgccccgg 840 gcgctaggcggaggctggag ggccgggcac gctggagggt tccgggcact cacgcgcctc 900 acgctttgctctctgcagct ccccgggatg cggtagcggc cgctgtgcgg aggccgcgaa 960 gcagctgcagccaccgccgc gcagatccac 990 7 1024 DNA Mus musculus promoter (-97)...(927)7 ttacaagaag accggccggg cagttccagg aatcgggggg cggggcgtac tggccgggta 60aatacccgcg cgcgcggcct ccgaggcggc tctaactctg actctacact cgcccgctcc 120tacgaccgct gtctctccgg gctcccggac gcccccttcc cggcccagct ctccgtcgag 180gtccctcgcc caggtccttt gcctgattcg cccaggagtg cgcctcatcg gcccggggag 240cagcgaagcc agagggggcg cacgcacggg gagccccttt gtagacttca cggctgccaa 300catctgggcg cagcgcgagc cactgctggg cgccgcctcg cctcggggac cataggaggc 360gcagccccaa ggccggagat ttcgcttcgg gactaggtag gaaggagggg cgcggtgtgg 420ggaagggtgg gggcatcggt ccagctcggg agcttttccc ggtttctcct ccccttcccg 480ggtcattccc ggtagggagg ggacgaggca gggggcagag cggatgagaa ccgaagatcc 540ctgattcccg tcatactcag actggggccc tcgggtttct cctgtcccct ctctcacata 600tctcgggttt ggcacccccc ttttttcgcc ctcgccactg aggacaccgg actgagaggc 660gccctgagcg tccctagggc tcttgtgtct ctccccatcc tggccgcgct cctggagacc 720caacttccac gcgcgagttt tctctgggcg tcctcctagg gcgggcaggg gaagagactg 780tctggggttg gccggcagtg accgaggaca gtcgagttcc gcgaggtggc tgggcctgag 840acacggtcta aagcggggca aaggggtgcc ccgggcgcta ggcggaggct ggagggccgg 900gcacgctgga gggttccggg cactcacgcg cctcacgctt tgctctctgc agctccccgg 960gatgcggtag cggccgctgt gcggaggccg cgaagcagct gcagccaccg ccgcgcagat 1020ccac 1024 8 201 DNA Mus musculus promoter (-97)...(104) 8 ttacaagaagaccggccggg cagttccagg aatcgggggg cggggcgtac tggccgggta 60 aatacccgcgcgcgcggcct ccgaggcggc tctaactctg actctacact cgcccgctcc 120 tacgaccgctgtctctccgg gctcccggac gcccccttcc cggcccagct ctccgtcgag 180 gtccctcgcccaggtccttt g 201 9 1014 DNA Mus musculus promoter (-87)...(927) 9accggccggg cagttccagg aatcgggggg cggggcgtac tggccgggta aatacccgcg 60cgcgcggcct ccgaggcggc tctaactctg actctacact cgcccgctcc tacgaccgct 120gtctctccgg gctcccggac gcccccttcc cggcccagct ctccgtcgag gtccctcgcc 180caggtccttt gcctgattcg cccaggagtg cgcctcatcg gcccggggag cagcgaagcc 240agagggggcg cacgcacggg gagccccttt gtagacttca cggctgccaa catctgggcg 300cagcgcgagc cactgctggg cgccgcctcg cctcggggac cataggaggc gcagccccaa 360ggccggagat ttcgcttcgg gactaggtag gaaggagggg cgcggtgtgg ggaagggtgg 420gggcatcggt ccagctcggg agcttttccc ggtttctcct ccccttcccg ggtcattccc 480ggtagggagg ggacgaggca gggggcagag cggatgagaa ccgaagatcc ctgattcccg 540tcatactcag actggggccc tcgggtttct cctgtcccct ctctcacata tctcgggttt 600ggcacccccc ttttttcgcc ctcgccactg aggacaccgg actgagaggc gccctgagcg 660tccctagggc tcttgtgtct ctccccatcc tggccgcgct cctggagacc caacttccac 720gcgcgagttt tctctgggcg tcctcctagg gcgggcaggg gaagagactg tctggggttg 780gccggcagtg accgaggaca gtcgagttcc gcgaggtggc tgggcctgag acacggtcta 840aagcggggca aaggggtgcc ccgggcgcta ggcggaggct ggagggccgg gcacgctgga 900gggttccggg cactcacgcg cctcacgctt tgctctctgc agctccccgg gatgcggtag 960cggccgctgt gcggaggccg cgaagcagct gcagccaccg ccgcgcagat ccac 1014 10 191DNA Mus musculus promoter (-87)...(104) 10 accggccggg cagttccaggaatcgggggg cggggcgtac tggccgggta aatacccgcg 60 cgcgcggcct ccgaggcggctctaactctg actctacact cgcccgctcc tacgaccgct 120 gtctctccgg gctcccggacgcccccttcc cggcccagct ctccgtcgag gtccctcgcc 180 caggtccttt g 191 11 433DNA Mus musculus promoter (-275)...(158) 11 caggaacgct cggccaaggctggcgccgcg cccgccggtc gggcagcctc gcgccgcgct 60 ttgtctccct ctcggtgagtctcggcgggt cctggaggcc ccagctccaa gcccgccctc 120 cgcagcccct ccctcgccctccgcgcacag cctttcagtg cagagtagtg actaaacatt 180 acaagaagac cggccgggcagttccaggaa tcggggggcg gggcgtactg gccgggtaaa 240 tacccgcgcg cgcggcctccgaggcggctc taactctgac tctacactcg cccgctccta 300 cgaccgctgt ctctccgggctcccggacgc ccccttcccg gcccagctct ccgtcgaggt 360 ccctcgccca ggtcctttgcctgattcgcc caggagtgcg cctcatcggc ccggggagca 420 gcgaagccag agg 433 121088 DNA Mus musculus promoter (-161)...(927) 12 gccctccgca gcccctccctcgccctccgc gcacagcctt tcagtgcaga gtagtgacta 60 aacattacaa gaagaccggccgggcagttc caggaatcgg ggggcggggc gtactggccg 120 ggtaaatacc cgcgcgcgcggcctccgagg cggctctaac tctgactcta cactcgcccg 180 ctcctacgac cgctgtctctccgggctccc ggacgccccc ttcccggccc agctctccgt 240 cgaggtccct cgcccaggtcctttgcctga ttcgcccagg agtgcgcctc atcggcccgg 300 ggagcagcga agccagagggggcgcacgca cggggagccc ctttgtagac ttcacggctg 360 ccaacatctg ggcgcagcgcgagccactgc tgggcgccgc ctcgcctcgg ggaccatagg 420 aggcgcagcc ccaaggccggagatttcgct tcgggactag gtaggaagga ggggcgcggt 480 gtggggaagg gtgggggcatcggtccagct cgggagcttt tcccggtttc tcctcccctt 540 cccgggtcat tcccggtagggaggggacga ggcagggggc agagcggatg agaaccgaag 600 atccctgatt cccgtcatactcagactggg gccctcgggt ttctcctgtc ccctctctca 660 catatctcgg gtttggcacccccctttttt cgccctcgcc actgaggaca ccggactgag 720 aggcgccctg agcgtccctagggctcttgt gtctctcccc atcctggccg cgctcctgga 780 gacccaactt ccacgcgcgagttttctctg ggcgtcctcc tagggcgggc aggggaagag 840 actgtctggg gttggccggcagtgaccgag gacagtcgag ttccgcgagg tggctgggcc 900 tgagacacgg tctaaagcggggcaaagggg tgccccgggc gctaggcgga ggctggaggg 960 ccgggcacgc tggagggttccgggcactca cgcgcctcac gctttgctct ctgcagctcc 1020 ccgggatgcg gtagcggccgctgtgcggag gccgcgaagc agctgcagcc accgccgcgc 1080 agatccac 1088 13 9 DNAMus musculus promoter (-74)...(-66) STAT-BINDING SITE AT -74 TO -66 13ttccaggaa 9 14 9 DNA Mus musculus mutation (-74)...(-66) 14 atcgacgat 915 27 DNA Artificial Sequence PRIMER 15 cagtagaatc cgctctcctg cagcttg 2716 27 DNA Artificial Sequence PRIMER 16 ctcgcttttg gagctgaagg tcttgag 2717 27 DNA Artificial Sequence PRIMER 17 cttcctacct agtcccgaag cgaaatc 2718 26 DNA Artificial Sequence PRIMER 18 cagatgttgg cagccgtgaa gtctac 2619 28 DNA Artificial Sequence PRIMER 19 gcgggcgagt gtagagtcag agttagag28 20 29 DNA Artificial Sequence PRIMER 20 cgattcctgg aactgcccggccggtcttc 29 21 29 DNA Artificial Sequence PRIMER 21 ctcagtgggctttctgacct gccctcttg 29 22 28 DNA Artificial Sequence PRIMER 22gactacacag agtagcttgg gctaggag 28 23 21 DNA Artificial Sequence SENSEPRIMER 23 catcgcgacg cccccgcctc t 21 24 22 DNA Artificial SequencePRIMER 24 gaaacccgag ggccccagtc tg 22 25 21 DNA Mus musculus promoter(-77)...(-57) 25 cagttccagg aatcgggggg c 21 26 21 DNA Mus musculuspromoter (0)...(0) MUTATIONS AT POSITIONS -74, -71, -69, & -66 26cagatcgacg attcgggggg c 21 27 26 DNA Artificial SequenceOLIGONUCLEOTIDES 27 gatcgaactg accgcccgcc gcccgt 26 28 30 DNA ArtificialSequence PRIMERS 28 gacgttccta aaagcatgca tgtcacccag 30 29 30 DNAArtificial Sequence PRIMERS 29 ggatctgcgc ggcggtggct gcagctgctt 30 30 9DNA Mus musculus promoter (-97)...(-89) STAT-BINDING SITE AT -97 TO -8930 ttacaagaa 9 31 9 DNA Artificial Sequence n=A, T, C, or G; consensusstat-binding element 31 ttnnnnnaa 9 32 289 DNA Mus musculus exon(1)...(289) Exon 1 of SOCS-3 (untranslated) 32 ctgactctac actcgcccgctcctacgacc gctgtctctc cgggctcccg gacgccccct 60 tcccggccca gctctccgtcgaggtccctc gcccaggtcc tttgcctgat tcgcccagga 120 gtgcgcctca tcggcccggggagcagcgaa gccagagggg gcgcacgcac ggggagcccc 180 tttgtagact tcacggctgccaacatctgg gcgcagcgcg agccactgct gggcgccgcc 240 tcgcctcggg gaccataggaggcgcagccc caaggccgga gatttcgct 289 33 564 DNA Mus musculus intron(290)...(853) Intron 1 of SOCS-3 33 tcgggactag gtaggaagga ggggcgcggtgtggggaagg gtgggggcat cggtccagct 60 cgggagcttt tcccggtttc tcctccccttcccgggtcat tcccggtagg gaggggacga 120 ggcagggggc agagcggatg agaaccgaagatccctgatt cccgtcatac tcagactggg 180 gccctcgggt ttctcctgtc ccctctctcacatatctcgg gtttggcacc cccctttttt 240 cgccctcgcc actgaggaca ccggactgagaggcgccctg agcgtcccta gggctcttgt 300 gtctctcccc atcctggccg cgctcctggagacccaactt ccacgcgcga gttttctctg 360 ggcgtcctcc tagggcgggc aggggaagagactgtctggg gttggccggc agtgaccgag 420 gacagtcgag ttccgcgagg tggctgggcctgagacacgg tctaaagcgg ggcaaagggg 480 tgccccgggc gctaggcgga ggctggagggccgggcacgc tggagggttc cgggcactca 540 cgcgcctcac gctttgctct ctgc 564 34180 DNA Mus musculus exon (854)...(1033) Partial exon 2 of SOCS-3 34agctccccgg gatgcggtag cggccgctgt gcggaggccg cgaagcagct gcagccaccg 60ccgcgcagat ccacgctggc tccgtgcgcc atggtcaccc acagcaagtt tcccgccgcc 120gggatgagcc gccccctgga caccagcctg cgcctcaaga ccttcagctc caaaagcgag 180 353665 DNA Mus musculus mutation (0)...(0) promoter sequence -2759 to +927with nucleotides -80 to -60 deleted 35 ccctgctggc cctgcagagg gaaggctctcctaagcatca tctatcagaa cgtcttcaaa 60 aaaaaaaaaa aaaaaaaaaa agcctctccagccaggctag ctctaacacc atttcttccc 120 cttcccctct ctcaaattca cttatctttttttttttttt tttttttgga tttttgagac 180 agggtttctc tgtatagccc tggttgtcctggaactcact ttgtacacca gcctggcctc 240 gaactcagag atccacctgc ctctgcctcctgagtgctgg gattaaaggc gtgcgccacc 300 acgcccggct aaattcactt atctatttaatgtatatagg gtataggctg cccttgaact 360 cacaaagatc tgcttgcttt gcttctggaatactaaaggt gtgtgctacc atcacaggga 420 ccaagattta ttttaattct gtatatgtgtgtgtgtgtgt gtgtgtatgg ggggtgcaca 480 tgagtacaga ttcccttgga ggcctggggtggcttaggac tggggttaca acagttgtga 540 cccatcctac ataggtcctg gcaccaacaccccccccccc cccccccgtc ttccagaagt 600 gcagcaggtg ttcttaactg ctgagccagcaatccagccc ctgacttccc tctcttactt 660 aagaagctat cacagtgtct cactgggtcacaatcatgac tagtccttgc tcatggccca 720 cagcctcttc cccactgtgg gttttgccccgcagctctgc cgccccagcg ctgcacccga 780 ggcctgacag agccaggcac gaagtcagggtttgtggaat ggatgaatga acttgactcg 840 tggcagagca ttgtaattta caaagcactttcccatccat taactccagg gctatttcct 900 aagagtcctc cctgtcctcc actgccctcggctcagaggc atacggtcaa ggcagtggct 960 ggggaacact ccctgaatga gatcaaggagggcttgttca cagagaaagg gagaatccat 1020 ttggggagcc tgagagtgac tcgaaggcaaggactgggcc tcacctgtgg gatctccatc 1080 tgtgagcatc cgctcatcag accagtgtgagatattttaa ataaggcccc taagcctctt 1140 gactactgga attgccaggg gcgggggacagatgggcacc catccctatt taacagataa 1200 caagactgag tccagagagg cagtgcacctgccctggtct ctcttagttc ctcagcatca 1260 gtggagcaga ttggacacag tgggccagaagggaagcagg cagccctccc tcctagccca 1320 agctactctg tgtagtcagt ttgccctcctactggtgtta caagaagcct gtggtatcca 1380 agagggcagg tcagaaagcc cactgagagcagacactgtg tgtcacttag ctggttctca 1440 ggtggctgcc acttcctgct gcctgttgcaaaactcgaca ctaggccttt atagatactc 1500 acgtgaccag gagtaaacaa cctttcacctcaatcacctg ctcttatcaa tactccctct 1560 ccaccccacc atcgggaaag ttcagacaccttaaaacgta gaggcaagag agggtccatt 1620 ctgacacctc agcgactttc aggcagtggctgaacccgtt acaacgctct gtggacagtc 1680 ctcctagtcg acattccttc tcaggtttgaccctgtcctg ggaagtgagg cttctctctc 1740 tgggttcccc actcctgttc ttgaataaggagccccacaa cctcttattc tctctataca 1800 gagcctggga aacagcaaaa ctcggctcgcctacaagact ccagcgcgcc ctctggtgga 1860 ctcgggggac gagcatggga tgagggtttctttcctctag ctcccccacc gcgccgagag 1920 tacctgggcg gacccacagt tcgccacgcaggttgggagg cccagatgag tgataaggta 1980 gtagttagct gctcctccca ccccactccccaaaggacat cagcacccac gtctgtcacc 2040 gaagaaccag gcaatgggcg gatgagctgaggccaggtag ctgcttctaa gtcagtgtct 2100 cctccacttc tggatctcac agcttcatcttttggacctg tctacaggta aatgtcgcgc 2160 atccccctcc tccacttcct aggtccccagtgggctggtg gctgaatggt cctacgtccc 2220 ttttggttgg cacgggatgc ttggaactgtacatgaggac ctcggggtgg cctgggtgca 2280 gagggagggg agcgtccccg cggggatcaaaagaaaggga aggggtgcca ggagggagcc 2340 tctcccggct ggcctcctag aactgcccgcgcgctcccat cgcgacgccc ccgcctctgc 2400 cagaaaccag ccttcttaga agggaggggggggaaagtgt gaatgagaag ttgggggcgg 2460 agcgcgcggg ggaggggccg ctgccaggaacgctcggcca aggctggcgc cgcgcccgcc 2520 ggtcgggcag cctcgcgccg cgctttgtctccctctcggt gagtctcggc gggtcctgga 2580 ggccccagct ccaagcccgc cctccgcagcccctccctcg ccctccgcgc acagcctttc 2640 agtgcagagt agtgactaaa cattacaagaagaccggccg gcggggcgta ctggccgggt 2700 aaatacccgc gcgcgcggcc tccgaggcggctctaactct gactctacac tcgcccgctc 2760 ctacgaccgc tgtctctccg ggctcccggacgcccccttc ccggcccagc tctccgtcga 2820 ggtccctcgc ccaggtcctt tgcctgattcgcccaggagt gcgcctcatc ggcccgggga 2880 gcagcgaagc cagagggggc gcacgcacggggagcccctt tgtagacttc acggctgcca 2940 acatctgggc gcagcgcgag ccactgctgggcgccgcctc gcctcgggga ccataggagg 3000 cgcagcccca aggccggaga tttcgcttcgggactaggta ggaaggaggg gcgcggtgtg 3060 gggaagggtg ggggcatcgg tccagctcgggagcttttcc cggtttctcc tccccttccc 3120 gggtcattcc cggtagggag gggacgaggcagggggcaga gcggatgaga accgaagatc 3180 cctgattccc gtcatactca gactggggccctcgggtttc tcctgtcccc tctctcacat 3240 atctcgggtt tggcaccccc cttttttcgccctcgccact gaggacaccg gactgagagg 3300 cgccctgagc gtccctaggg ctcttgtgtctctccccatc ctggccgcgc tcctggagac 3360 ccaacttcca cgcgcgagtt ttctctgggcgtcctcctag ggcgggcagg ggaagagact 3420 gtctggggtt ggccggcagt gaccgaggacagtcgagttc cgcgaggtgg ctgggcctga 3480 gacacggtct aaagcggggc aaaggggtgccccgggcgct aggcggaggc tggagggccg 3540 ggcacgctgg agggttccgg gcactcacgcgcctcacgct ttgctctctg cagctccccg 3600 ggatgcggta gcggccgctg tgcggaggccgcgaagcagc tgcagccacc gccgcgcaga 3660 tccac 3665 36 3646 DNA Musmusculus mutation (0)...(0) promoter sequence -2759 to +927 withnucleotides -101 to -62 deleted 36 ccctgctggc cctgcagagg gaaggctctcctaagcatca tctatcagaa cgtcttcaaa 60 aaaaaaaaaa aaaaaaaaaa agcctctccagccaggctag ctctaacacc atttcttccc 120 cttcccctct ctcaaattca cttatctttttttttttttt tttttttgga tttttgagac 180 agggtttctc tgtatagccc tggttgtcctggaactcact ttgtacacca gcctggcctc 240 gaactcagag atccacctgc ctctgcctcctgagtgctgg gattaaaggc gtgcgccacc 300 acgcccggct aaattcactt atctatttaatgtatatagg gtataggctg cccttgaact 360 cacaaagatc tgcttgcttt gcttctggaatactaaaggt gtgtgctacc atcacaggga 420 ccaagattta ttttaattct gtatatgtgtgtgtgtgtgt gtgtgtatgg ggggtgcaca 480 tgagtacaga ttcccttgga ggcctggggtggcttaggac tggggttaca acagttgtga 540 cccatcctac ataggtcctg gcaccaacaccccccccccc cccccccgtc ttccagaagt 600 gcagcaggtg ttcttaactg ctgagccagcaatccagccc ctgacttccc tctcttactt 660 aagaagctat cacagtgtct cactgggtcacaatcatgac tagtccttgc tcatggccca 720 cagcctcttc cccactgtgg gttttgccccgcagctctgc cgccccagcg ctgcacccga 780 ggcctgacag agccaggcac gaagtcagggtttgtggaat ggatgaatga acttgactcg 840 tggcagagca ttgtaattta caaagcactttcccatccat taactccagg gctatttcct 900 aagagtcctc cctgtcctcc actgccctcggctcagaggc atacggtcaa ggcagtggct 960 ggggaacact ccctgaatga gatcaaggagggcttgttca cagagaaagg gagaatccat 1020 ttggggagcc tgagagtgac tcgaaggcaaggactgggcc tcacctgtgg gatctccatc 1080 tgtgagcatc cgctcatcag accagtgtgagatattttaa ataaggcccc taagcctctt 1140 gactactgga attgccaggg gcgggggacagatgggcacc catccctatt taacagataa 1200 caagactgag tccagagagg cagtgcacctgccctggtct ctcttagttc ctcagcatca 1260 gtggagcaga ttggacacag tgggccagaagggaagcagg cagccctccc tcctagccca 1320 agctactctg tgtagtcagt ttgccctcctactggtgtta caagaagcct gtggtatcca 1380 agagggcagg tcagaaagcc cactgagagcagacactgtg tgtcacttag ctggttctca 1440 ggtggctgcc acttcctgct gcctgttgcaaaactcgaca ctaggccttt atagatactc 1500 acgtgaccag gagtaaacaa cctttcacctcaatcacctg ctcttatcaa tactccctct 1560 ccaccccacc atcgggaaag ttcagacaccttaaaacgta gaggcaagag agggtccatt 1620 ctgacacctc agcgactttc aggcagtggctgaacccgtt acaacgctct gtggacagtc 1680 ctcctagtcg acattccttc tcaggtttgaccctgtcctg ggaagtgagg cttctctctc 1740 tgggttcccc actcctgttc ttgaataaggagccccacaa cctcttattc tctctataca 1800 gagcctggga aacagcaaaa ctcggctcgcctacaagact ccagcgcgcc ctctggtgga 1860 ctcgggggac gagcatggga tgagggtttctttcctctag ctcccccacc gcgccgagag 1920 tacctgggcg gacccacagt tcgccacgcaggttgggagg cccagatgag tgataaggta 1980 gtagttagct gctcctccca ccccactccccaaaggacat cagcacccac gtctgtcacc 2040 gaagaaccag gcaatgggcg gatgagctgaggccaggtag ctgcttctaa gtcagtgtct 2100 cctccacttc tggatctcac agcttcatcttttggacctg tctacaggta aatgtcgcgc 2160 atccccctcc tccacttcct aggtccccagtgggctggtg gctgaatggt cctacgtccc 2220 ttttggttgg cacgggatgc ttggaactgtacatgaggac ctcggggtgg cctgggtgca 2280 gagggagggg agcgtccccg cggggatcaaaagaaaggga aggggtgcca ggagggagcc 2340 tctcccggct ggcctcctag aactgcccgcgcgctcccat cgcgacgccc ccgcctctgc 2400 cagaaaccag ccttcttaga agggaggggggggaaagtgt gaatgagaag ttgggggcgg 2460 agcgcgcggg ggaggggccg ctgccaggaacgctcggcca aggctggcgc cgcgcccgcc 2520 ggtcgggcag cctcgcgccg cgctttgtctccctctcggt gagtctcggc gggtcctgga 2580 ggccccagct ccaagcccgc cctccgcagcccctccctcg ccctccgcgc acagcctttc 2640 agtgcagagt agtgactagg ggcggggcgtactggccggg taaatacccg cgcgcgcggc 2700 ctccgaggcg gctctaactc tgactctacactcgcccgct cctacgaccg ctgtctctcc 2760 gggctcccgg acgccccctt cccggcccagctctccgtcg aggtccctcg cccaggtcct 2820 ttgcctgatt cgcccaggag tgcgcctcatcggcccgggg agcagcgaag ccagaggggg 2880 cgcacgcacg gggagcccct ttgtagacttcacggctgcc aacatctggg cgcagcgcga 2940 gccactgctg ggcgccgcct cgcctcggggaccataggag gcgcagcccc aaggccggag 3000 atttcgcttc gggactaggt aggaaggaggggcgcggtgt ggggaagggt gggggcatcg 3060 gtccagctcg ggagcttttc ccggtttctcctccccttcc cgggtcattc ccggtaggga 3120 ggggacgagg cagggggcag agcggatgagaaccgaagat ccctgattcc cgtcatactc 3180 agactggggc cctcgggttt ctcctgtcccctctctcaca tatctcgggt ttggcacccc 3240 ccttttttcg ccctcgccac tgaggacaccggactgagag gcgccctgag cgtccctagg 3300 gctcttgtgt ctctccccat cctggccgcgctcctggaga cccaacttcc acgcgcgagt 3360 tttctctggg cgtcctccta gggcgggcaggggaagagac tgtctggggt tggccggcag 3420 tgaccgagga cagtcgagtt ccgcgaggtggctgggcctg agacacggtc taaagcgggg 3480 caaaggggtg ccccgggcgc taggcggaggctggagggcc gggcacgctg gagggttccg 3540 ggcactcacg cgcctcacgc tttgctctctgcagctcccc gggatgcggt agcggccgct 3600 gtgcggaggc cgcgaagcag ctgcagccaccgccgcgcag atccac 3646 37 3686 DNA Mus musculus mutation (0)...(0)promoter sequence -2759 to +927 with nucleotide at position -74 mutatedfrom T to A, nucleotide at position -71 mutated from C to G, nucleotideat position -69 mutated from G to C, and nucleotide at position -66mutated from A to T 37 ccctgctggc cctgcagagg gaaggctctc ctaagcatcatctatcagaa cgtcttcaaa 60 aaaaaaaaaa aaaaaaaaaa agcctctcca gccaggctagctctaacacc atttcttccc 120 cttcccctct ctcaaattca cttatctttt tttttttttttttttttgga tttttgagac 180 agggtttctc tgtatagccc tggttgtcct ggaactcactttgtacacca gcctggcctc 240 gaactcagag atccacctgc ctctgcctcc tgagtgctgggattaaaggc gtgcgccacc 300 acgcccggct aaattcactt atctatttaa tgtatatagggtataggctg cccttgaact 360 cacaaagatc tgcttgcttt gcttctggaa tactaaaggtgtgtgctacc atcacaggga 420 ccaagattta ttttaattct gtatatgtgt gtgtgtgtgtgtgtgtatgg ggggtgcaca 480 tgagtacaga ttcccttgga ggcctggggt ggcttaggactggggttaca acagttgtga 540 cccatcctac ataggtcctg gcaccaacac cccccccccccccccccgtc ttccagaagt 600 gcagcaggtg ttcttaactg ctgagccagc aatccagcccctgacttccc tctcttactt 660 aagaagctat cacagtgtct cactgggtca caatcatgactagtccttgc tcatggccca 720 cagcctcttc cccactgtgg gttttgcccc gcagctctgccgccccagcg ctgcacccga 780 ggcctgacag agccaggcac gaagtcaggg tttgtggaatggatgaatga acttgactcg 840 tggcagagca ttgtaattta caaagcactt tcccatccattaactccagg gctatttcct 900 aagagtcctc cctgtcctcc actgccctcg gctcagaggcatacggtcaa ggcagtggct 960 ggggaacact ccctgaatga gatcaaggag ggcttgttcacagagaaagg gagaatccat 1020 ttggggagcc tgagagtgac tcgaaggcaa ggactgggcctcacctgtgg gatctccatc 1080 tgtgagcatc cgctcatcag accagtgtga gatattttaaataaggcccc taagcctctt 1140 gactactgga attgccaggg gcgggggaca gatgggcacccatccctatt taacagataa 1200 caagactgag tccagagagg cagtgcacct gccctggtctctcttagttc ctcagcatca 1260 gtggagcaga ttggacacag tgggccagaa gggaagcaggcagccctccc tcctagccca 1320 agctactctg tgtagtcagt ttgccctcct actggtgttacaagaagcct gtggtatcca 1380 agagggcagg tcagaaagcc cactgagagc agacactgtgtgtcacttag ctggttctca 1440 ggtggctgcc acttcctgct gcctgttgca aaactcgacactaggccttt atagatactc 1500 acgtgaccag gagtaaacaa cctttcacct caatcacctgctcttatcaa tactccctct 1560 ccaccccacc atcgggaaag ttcagacacc ttaaaacgtagaggcaagag agggtccatt 1620 ctgacacctc agcgactttc aggcagtggc tgaacccgttacaacgctct gtggacagtc 1680 ctcctagtcg acattccttc tcaggtttga ccctgtcctgggaagtgagg cttctctctc 1740 tgggttcccc actcctgttc ttgaataagg agccccacaacctcttattc tctctataca 1800 gagcctggga aacagcaaaa ctcggctcgc ctacaagactccagcgcgcc ctctggtgga 1860 ctcgggggac gagcatggga tgagggtttc tttcctctagctcccccacc gcgccgagag 1920 tacctgggcg gacccacagt tcgccacgca ggttgggaggcccagatgag tgataaggta 1980 gtagttagct gctcctccca ccccactccc caaaggacatcagcacccac gtctgtcacc 2040 gaagaaccag gcaatgggcg gatgagctga ggccaggtagctgcttctaa gtcagtgtct 2100 cctccacttc tggatctcac agcttcatct tttggacctgtctacaggta aatgtcgcgc 2160 atccccctcc tccacttcct aggtccccag tgggctggtggctgaatggt cctacgtccc 2220 ttttggttgg cacgggatgc ttggaactgt acatgaggacctcggggtgg cctgggtgca 2280 gagggagggg agcgtccccg cggggatcaa aagaaagggaaggggtgcca ggagggagcc 2340 tctcccggct ggcctcctag aactgcccgc gcgctcccatcgcgacgccc ccgcctctgc 2400 cagaaaccag ccttcttaga agggaggggg gggaaagtgtgaatgagaag ttgggggcgg 2460 agcgcgcggg ggaggggccg ctgccaggaa cgctcggccaaggctggcgc cgcgcccgcc 2520 ggtcgggcag cctcgcgccg cgctttgtct ccctctcggtgagtctcggc gggtcctgga 2580 ggccccagct ccaagcccgc cctccgcagc ccctccctcgccctccgcgc acagcctttc 2640 agtgcagagt agtgactaaa cattacaaga agaccggccgggcagatcga cgattcgggg 2700 ggcggggcgt actggccggg taaatacccg cgcgcgcggcctccgaggcg gctctaactc 2760 tgactctaca ctcgcccgct cctacgaccg ctgtctctccgggctcccgg acgccccctt 2820 cccggcccag ctctccgtcg aggtccctcg cccaggtcctttgcctgatt cgcccaggag 2880 tgcgcctcat cggcccgggg agcagcgaag ccagagggggcgcacgcacg gggagcccct 2940 ttgtagactt cacggctgcc aacatctggg cgcagcgcgagccactgctg ggcgccgcct 3000 cgcctcgggg accataggag gcgcagcccc aaggccggagatttcgcttc gggactaggt 3060 aggaaggagg ggcgcggtgt ggggaagggt gggggcatcggtccagctcg ggagcttttc 3120 ccggtttctc ctccccttcc cgggtcattc ccggtagggaggggacgagg cagggggcag 3180 agcggatgag aaccgaagat ccctgattcc cgtcatactcagactggggc cctcgggttt 3240 ctcctgtccc ctctctcaca tatctcgggt ttggcaccccccttttttcg ccctcgccac 3300 tgaggacacc ggactgagag gcgccctgag cgtccctagggctcttgtgt ctctccccat 3360 cctggccgcg ctcctggaga cccaacttcc acgcgcgagttttctctggg cgtcctccta 3420 gggcgggcag gggaagagac tgtctggggt tggccggcagtgaccgagga cagtcgagtt 3480 ccgcgaggtg gctgggcctg agacacggtc taaagcggggcaaaggggtg ccccgggcgc 3540 taggcggagg ctggagggcc gggcacgctg gagggttccgggcactcacg cgcctcacgc 3600 tttgctctct gcagctcccc gggatgcggt agcggccgctgtgcggaggc cgcgaagcag 3660 ctgcagccac cgccgcgcag atccac 3686 38 2044 DNAMus musculus promoter (-2759)...(-716) 38 ccctgctggc cctgcagagggaaggctctc ctaagcatca tctatcagaa cgtcttcaaa 60 aaaaaaaaaa aaaaaaaaaaagcctctcca gccaggctag ctctaacacc atttcttccc 120 cttcccctct ctcaaattcacttatctttt tttttttttt tttttttgga tttttgagac 180 agggtttctc tgtatagccctggttgtcct ggaactcact ttgtacacca gcctggcctc 240 gaactcagag atccacctgcctctgcctcc tgagtgctgg gattaaaggc gtgcgccacc 300 acgcccggct aaattcacttatctatttaa tgtatatagg gtataggctg cccttgaact 360 cacaaagatc tgcttgctttgcttctggaa tactaaaggt gtgtgctacc atcacaggga 420 ccaagattta ttttaattctgtatatgtgt gtgtgtgtgt gtgtgtatgg ggggtgcaca 480 tgagtacaga ttcccttggaggcctggggt ggcttaggac tggggttaca acagttgtga 540 cccatcctac ataggtcctggcaccaacac cccccccccc cccccccgtc ttccagaagt 600 gcagcaggtg ttcttaactgctgagccagc aatccagccc ctgacttccc tctcttactt 660 aagaagctat cacagtgtctcactgggtca caatcatgac tagtccttgc tcatggccca 720 cagcctcttc cccactgtgggttttgcccc gcagctctgc cgccccagcg ctgcacccga 780 ggcctgacag agccaggcacgaagtcaggg tttgtggaat ggatgaatga acttgactcg 840 tggcagagca ttgtaatttacaaagcactt tcccatccat taactccagg gctatttcct 900 aagagtcctc cctgtcctccactgccctcg gctcagaggc atacggtcaa ggcagtggct 960 ggggaacact ccctgaatgagatcaaggag ggcttgttca cagagaaagg gagaatccat 1020 ttggggagcc tgagagtgactcgaaggcaa ggactgggcc tcacctgtgg gatctccatc 1080 tgtgagcatc cgctcatcagaccagtgtga gatattttaa ataaggcccc taagcctctt 1140 gactactgga attgccaggggcgggggaca gatgggcacc catccctatt taacagataa 1200 caagactgag tccagagaggcagtgcacct gccctggtct ctcttagttc ctcagcatca 1260 gtggagcaga ttggacacagtgggccagaa gggaagcagg cagccctccc tcctagccca 1320 agctactctg tgtagtcagtttgccctcct actggtgtta caagaagcct gtggtatcca 1380 agagggcagg tcagaaagcccactgagagc agacactgtg tgtcacttag ctggttctca 1440 ggtggctgcc acttcctgctgcctgttgca aaactcgaca ctaggccttt atagatactc 1500 acgtgaccag gagtaaacaacctttcacct caatcacctg ctcttatcaa tactccctct 1560 ccaccccacc atcgggaaagttcagacacc ttaaaacgta gaggcaagag agggtccatt 1620 ctgacacctc agcgactttcaggcagtggc tgaacccgtt acaacgctct gtggacagtc 1680 ctcctagtcg acattccttctcaggtttga ccctgtcctg ggaagtgagg cttctctctc 1740 tgggttcccc actcctgttcttgaataagg agccccacaa cctcttattc tctctataca 1800 gagcctggga aacagcaaaactcggctcgc ctacaagact ccagcgcgcc ctctggtgga 1860 ctcgggggac gagcatgggatgagggtttc tttcctctag ctcccccacc gcgccgagag 1920 tacctgggcg gacccacagttcgccacgca ggttgggagg cccagatgag tgataaggta 1980 gtagttagct gctcctcccaccccactccc caaaggacat cagcacccac gtctgtcacc 2040 gaag 2044 39 433 DNAMus musculus promoter (-275)...(158) 39 caggaacgct cggccaaggc tggcgccgcgcccgccggtc gggcagcctc gcgccgcgct 60 ttgtctccct ctcggtgagt ctcggcgggtcctggaggcc ccagctccaa gcccgccctc 120 cgcagcccct ccctcgccct ccgcgcacagcctttcagtg cagagtagtg actaaacatt 180 acaagaagac cggccgggca gttccaggaatcggggggcg gggcgtactg gccgggtaaa 240 tacccgcgcg cgcggcctcc gaggcggctctaactctgac tctacactcg cccgctccta 300 cgaccgctgt ctctccgggc tcccggacgcccccttcccg gcccagctct ccgtcgaggt 360 ccctcgccca ggtcctttgc ctgattcgcccaggagtgcg cctcatcggc ccggggagca 420 gcgaagccag agg 433

We claim:
 1. A nucleic acid construct, comprising a murine SOCS-3promoter having a nucleotide sequence SEQ ID NO: 1, or an operativefragment of SEQ ID NO: 1 having promoter activity that is inducible byLIF, IL-6, and IL-11.
 2. The nucleic acid construct of claim 1, furthercomprising said SOCS-3 promoter operatively linked, in a transcriptionalunit, to a DNA sequence encoding a protein of interest.
 3. The nucleicacid construct of claim 2, wherein the protein of interest is a SOCS-3protein, or a functional fragment thereof.
 4. The nucleic acid constructof claim 2, wherein said SOCS-3 promoter is operatively linked to a DNAsequence encoding a reporter protein.
 5. The nucleic acid construct ofclaim 4, wherein the reporter protein is a fluorescent or light-emittingprotein.
 6. The nucleic acid construct of claim 5, wherein the encodedfluorescent or light-emitting protein is a green fluorescent protein,yellow fluorescent protein, blue fluorescent protein, phycobiliprotein,luciferase, or apoaequorin.
 7. The nucleic acid construct of claim 2,further comprising at least one insulator element flanking saidtranscriptional unit.
 8. The nucleic acid construct of claim 7, whereinsaid at least one insulator element is a chicken β-globin insulatorelement.
 9. A kit for genetically modifying a vertebrate cell,containing a polynucleotide comprising a murine SOCS-3 promoter sequencehaving a nucleotide sequence SEQ ID NO: 1, or an operative fragment ofSEQ ID NO: 1 having promoter activity that is inducible by LIF, IL-6,and IL-11, and a suitable storage means for said polynucleotide.
 10. Thekit of claim 9, further comprising said SOCS-3 promoter operativelylinked, in a transcriptional unit, to a DNA sequence encoding a proteinof interest.
 11. The kit of claim 10, wherein the protein of interest isa SOCS-3 protein, or a functional fragment thereof.
 12. The kit of claim10, wherein said SOCS-3 promoter is operatively linked to a DNA sequenceencoding a reporter protein.
 13. The kit of claim 12, wherein theencoded reporter protein is a fluorescent or light-emitting protein. 14.The kit of claim 13, wherein the encoded fluorescent or light-emittingprotein is a green fluorescent protein, yellow fluorescent protein, bluefluorescent protein, phycobiliprotein, luciferase, or apoaequorin. 15.The kit of claim 10, wherein said polynucleotide further comprises atleast one insulator element flanking said transcriptional unit.
 16. Thekit of claim 15, wherein said at least one insulator element is achicken β-globin insulator element.
 17. The kit of claim 9, furthercomprising a transfecting agent.
 18. The kit of claim 17, wherein thetransfecting agent is a liposome, viral vector, transferrin-polylysineenhanced viral vector, retroviral vector, lentiviral vector, or uptakeenhancing DNA segment, or a mixture of any of these.
 19. The kit ofclaim 17, wherein the transfecting agent comprises a retroviral vector,adenoviral vector, transferrin-polylysine enhanced adenoviral vector,human immunodeficiency virus vector, lentiviral vector, Moloney murineleukemia virus-derived vector, mumps vector, a DNA segment thatfacilitates polynucleotide uptake by and release into the cytoplasm ofvertebrate cells, or comprises an operative fragment of or mixture ofany of these.
 20. The kit of claim 17, wherein the transfecting agentcomprises an adenovirus vector having endosomal lytic activity, and thepolynucleotide is operatively linked to the vector.
 21. The kit of claim17, wherein the transfecting agent comprises a lipid transfecting agent.22. A nucleic acid construct, comprising a murine SOCS-3 promoter havinga nucleotide sequence (SEQ ID NO: 1), (SEQ ID NO: 2), (SEQ ID NO: 3),(SEQ ID NO: 4), (SEQ ID NO: 5), (SEQ ID NO: 6), (SEQ ID NO: 7), (SEQ IDNO: 8), (SEQ ID NO: 9), (SEQ ID NO: 10), (SEQ ID NO: 11), (SEQ ID NO:12), (SEQ ID NO: 35), (SEQ ID NO: 36), (SEQ ID NO: 37), or (SEQ ID NO:39).
 23. The nucleic acid construct of claim 22, further comprising saidSOCS-3 promoter operatively linked, in a transcriptional unit, to a DNAsequence encoding a protein of interest.
 24. The nucleic acid constructof claim 23, wherein the protein of interest is a SOCS-3 protein. 25.The nucleic acid construct of claim 23, wherein said SOCS-3 promoter isoperatively linked to a DNA sequence encoding a reporter protein. 26.The nucleic acid construct of claim 25, wherein the reporter protein isa fluorescent or light-emitting protein.
 27. The nucleic acid constructof claim 26, wherein the encoded fluorescent or light-emitting proteinis a green fluorescent protein, yellow fluorescent protein, bluefluorescent protein, phycobiliprotein, luciferase, or apoaequorin. 28.The nucleic acid construct of claim 23, further comprising at least oneinsulator element flanking said transcriptional unit.
 29. The nucleicacid construct of claim 28, wherein said at least one insulator elementis a chicken β-globin insulator element.
 30. A kit for geneticallymodifying a vertebrate cell, containing a polynucleotide comprising amurine SOCS-3 promoter sequence having a nucleotide sequence SEQ ID NO:1, (SEQ ID NO: 2), (SEQ ID NO: 3), (SEQ ID NO: 4), (SEQ ID NO: 5), (SEQID NO: 6), (SEQ ID NO: 7), (SEQ ID NO: 8), (SEQ ID NO: 9), (SEQ ID NO:10), (SEQ ID NO: 11), or (SEQ ID NO: 12), (SEQ ID NO: 35), (SEQ ID NO:36), (SEQ ID NO 37), or (SEQ ID NO: 39), and a suitable storage meansfor said polynucleotide.
 31. The kit of claim 30, further comprisingsaid SOCS-3 promoter operatively linked, in a transcriptional unit, to aDNA sequence encoding a protein of interest.
 32. The kit of claim 31,wherein the protein of interest is a SOCS-3 protein.
 33. The kit ofclaim 31, wherein said SOCS-3 promoter is operatively linked to a DNAsequence encoding a reporter protein.
 34. The kit of claim 33, whereinthe encoded reporter protein is a fluorescent or light-emitting protein.35. The kit of claim 34, wherein the encoded fluorescent orlight-emitting protein is a green fluorescent protein, yellowfluorescent protein, blue fluorescent protein, phycobiliprotein,luciferase, or apoaequorin.
 36. The kit of claim 31, wherein saidpolynucleotide further comprises at least one insulator element flankingsaid transcriptional unit.
 37. The kit of claim 36, wherein said atleast one insulator element is a chicken β-globin insulator element. 38.The kit of claim 30, further comprising a transfecting agent.
 39. Thekit of claim 38, wherein the transfecting agent is a liposome, viralvector, transferrin-polylysine enhanced viral vector, retroviral vector,lentiviral vector, or uptake enhancing DNA segment, or a mixture of anyof these.
 40. The kit of claim 38, wherein the transfecting agentcomprises a retroviral vector, adenoviral vector, transferrin-polylysineenhanced adenoviral vector, human immunodeficiency virus vector,lentiviral vector, Moloney murine leukemia virus-derived vector, mumpsvector, a DNA segment that facilitates polynucleotide uptake by andrelease into the cytoplasm of vertebrate cells, or comprises anoperative fragment of or mixture of any of these.
 41. The kit of claim38, wherein the transfecting agent comprises an adenovirus vector havingendosomal lytic activity, and the polynucleotide is operatively linkedto the vector.
 42. The kit of claim 38, wherein the transfecting agentcomprises a lipid transfecting agent.